Chumash people were the first to inhabit what is now called Thousand Oaks, settling there over 10,000 years ago. It was home to two major villages: Sap'wi ("House of the Deer") and Satwiwa ("The Bluffs"). Sap'wi is now by the Chumash Interpretive Center which is home to multiple 2,000 year-old pictographs.Satwiwa is the home of the Native American Indian Culture Center which sits at the foothills of Mount Boney in Newbury Park, a sacred mountain to the Chumash.

A smaller village, Yitimasɨh, was located where Wildwood Elementary School sits today. The area surrounding Wildwood Regional Park has been inhabited by the Chumash for thousands of years. Some of the artifacts discovered in Wildwood include stone tools, shell beads and arrowheads. Another small Chumash settlement, known as Šihaw (Ven-632i), was located where Lang Ranch sits today. A cave containing several swordfish and cupules pictographs is located here. Two other villages were located by today's Ventu Park Road in Newbury Park. These were populated 2,000 years ago and had a population of 100–200 in each village. Other villages included Lalimanuc (Lalimanux) and Kayɨwɨš (Kayiwish) by Conejo Grade.

The Chumash also had several summer encampments, including one located where Thousand Oaks Civic Arts Plaza currently stands, known as Ipuc (Ven-654). Another summer encampment was located at the current location of Los Robles Hospital.

Each village was ruled by a chief or several chieftains, who often traveled between villages to discuss matters of common interest. A council of elders directed village life and organized events. Most villages had a cemetery, gaming field, a sweat house, and a place for ceremonies. Locally discovered tribal artifacts are at display at Satwiwa Native American Indian Culture Center and the Chumash Indian Museum.

The region's recorded history dates to 1542, when Spanish explorer Juan Rodriguez Cabrillo landed at Point Mugu and claimed the land for Spain. The Battle of Triunfo, which took place by Triunfo Creek, was waged over land between native Chumash and the Spanish newcomers.

From 1804 to 1848, Thousand Oaks was part of Alta California, which originally was a Spanish polity in North America. It was the Spaniards who first named it Conejo Valley, or Valley of Rabbits. The Spaniards and indigenous Chumash clashed numerous times in disputes over land. Conejo Valley was given the name El Rancho Conejo in 1803. This year, Jose Polanco and Ignacio Rodriguez were granted El Rancho Conejo by Governor José Joaquín de Arrillaga of Alta California. The land contained 48,671.56 acres. El Conejo was just one of two land grants in what became Ventura County, the other being Rancho Simi.

As a result of the Mexican War of Independence in 1822, Alta California became a Mexican territory. In 1822, Captain José de la Guerra y Noriega filed Conejo Valley as part of the Mexican land grant. It remained a part of Mexico until the short-lived California Republic was established in 1846. It became a part of the U.S. after California gained statehood in 1850. The valley was now known as Rancho El Conejo. The ranch period began when the de la Guerra family sold thousands of acres through the 1860s and early 1870s.

Two men owned most of Conejo Valley in the 1870s: John Edwards, who came from Wales in 1849, and Howard Mills, who came from Minnesota in 1870. While Edwards owned most of present-day Thousand Oaks and Newbury Park, Mills owned most of Westlake Village and Hidden Valley. Edwards' home was located on an acre of land where The Oaks Mall currently is located, while Mills built his home where Westlake Lake sits today. The third person to buy former Rancho El Conejo land was Egbert Starr Newbury. He bought 2,259 acres of land here in 1874, land which stretched from Old Town Thousand Oaks and into today's Newbury Park. He later established the valley's first post office in 1875: Newbury Park Post Office. When the Conejo Valley School District was established in March 1877, there were 126 residents living in Conejo Valley.

In the late 19th century, Newbury Park was on the stagecoach route between Los Angeles and Santa Barbara. The Stagecoach Inn (Grand Union Hotel) was built in 1876, and is now a California Historical Landmark and museum.^{[citation needed]}

Thousand Oaks was home to a Norwegian community in the late 1890s and early 1900s, known as Norwegian Colony. Norwegian settlers were among the first to settle in Conejo Valley. The Norwegian Colony was located at today's intersection of Moorpark- and Olsen Roads, now home to California Lutheran University and surrounding areas. The Norwegian Colony constituted of over 650 acres and stretched from Mount Clef Ridge to Avenida de Los Arboles. The son of Norwegian immigrants donated his ranch to California Lutheran College in the 1950s. California Lutheran University is now home to the Scandinavian American Cultural and Historical Foundation and the Scandinavian Festival.^{[citation needed]}

Many place names are named after Norwegian immigrants such as the Olsen and Pedersen families. The first Norwegians came from the village of Stranda by Storfjorden. Ole Anderson bought 199 acres here, while Lars Pederson owned 111 acres. Other Norwegian pioneers also included Ole Nilsen, George Hansen and Nils Olsen. A major contribution was the construction of the handmade Norwegian Grade in 1911, a mile-long road leading from Thousand Oaks to Santa Rosa Valley.

With no doctors or hospitals nearby, the Norwegian Colony was short-lived. The Olsen family lost seven of their ten children, while Ole Anderson, Lars Pederson, and George Hansen all died in 1901 due to a diphtheria epidemic.

Newbury Park was a more established community than Thousand Oaks at the turn of the 20th century. A few lots existed early in the 1900s, wedged between Borchard land on the south and Friedrich land on the north. The Janss family, developers of Southern California subdivisions, purchased 10,000 acres (40 km²) in the early 20th century. They eventually created plans for a "total community", and the name remains prominently featured in the city. Despite early aspirations, no large subdivisions were developed until the 1920s. The development was slow and hampered even more under the Great Depression of the 1930s. Besides agriculture, the movie industry became an important industry in the 1920s and 1930s.

Between 1950 and 1970, Conejo Valley experienced a population boom, and increased its population from 3,000 to 30,000 residents. From 3,500 residents in 1957, Thousand Oaks had over 103,000 inhabitants by 1989. While ranching and agriculture were the dominant industries until the 1950s, a number of new businesses appeared throughout the 1960s and 1970s. Particularly many high-tech firms moved to Thousand Oaks in the '60s and '70s. Packard Bell and Technology Instrument Company were two high-technology businesses that moved into the Newbury Park industrial park in the 1960s. Other companies that followed included Westinghouse Astroelectronics Laboratory, Semtech Corporation, Purolator Inc., and Westland Plastics.

Jungleland USA put Thousand Oaks on the map in the 1920s and helped attract Hollywood producers to the city. Hundreds of movies have been filmed in Thousand Oaks. Some of the first films to be made here were The Birth of a Nation (1915) at Jungleland USA and Roaring Ranch (1930) at the Stagecoach Inn.Thousand Oaks Boulevard was featured in the "Walls of Jericho" scenes in the film It Happened One Night (1934). A western village was erected at California Lutheran University for the filming of Welcome to Hard Times (1967), while Elvis Presley and John Wayne starred in several westerns made in Wildwood Regional Park. A nearby road, Flaming Star Avenue, is named after the film Flaming Star (1960) starring Elvis Presley, which was filmed here. Other movies filmed in the valley included Lassie Come Home (1943), To the Shores of Iwo Jima (1945) and The Dukes of Hazzard (1979–85). Dean Martin and Jerry Lewis visited Thousand Oaks for the filming of Hollywood or Bust (1956), which included a scene filmed on Live Oak Street.

Movie actor Joel McCrea, who had been advised by Will Rogers to buy land in the area, raised his family on a 3,000-acre (1,200 ha) ranch he had acquired in the early 1930s. Numerous celebrities later joined McCrea and relocated to the Conejo Valley, including Dean Martin, Bob Hope, Roy Rogers, Strother Martin, Virginia Mayo, Michael O'Shea, Ben Johnson, Slim Pickens, Ronald Colman, George Brent, Eve Arden, Alan Ladd, Richard Widmark, Charles Martin Smith, and Bing- and Kurt Russell.

While the city was home to 1,700 businesses in 1970, Thousand Oaks had 11,000 businesses in town by 1988.

The world's largest independent biotechnology company, Amgen, was established in Newbury Park in 1980.

Louis Goebel of New York bought five lots off Ventura Boulevard (today's Thousand Oaks Boulevard) in 1925. He worked for the Universal film studio, and decided to create his own film industry zoo after the closure of Universal Zoo in the mid-1920s. He established Goebel's Lion Farm in 1926, situated where Thousand Oaks Civic Arts Plaza is located today. Goebel began with five lions and seven malamute dogs, but he soon acquired new animals such as giraffes, camels, hippos, monkeys, tigers, gorillas, seals and other exotic animals.^{[citation needed]}

It became home to several animals used for Leo the Lion MGM logo. There were held public animal shows, which drew thousands of spectators from throughout California. The animals from the park have been used in many movies and TV series, including many of the Tarzan films; The Adventures of Robin Hood (1938), which used the site as a location, and Doctor Doolittle (1967). Goebel himself camped by the filming site of Tarzan, the Ape Man (1932) by Lake Sherwood to watch his lions during filming.

It became one of Southern California's most popular tourists attractions in the 1940s and 1950s, when the 170-acre park offered shows, lion training, elephant rides, train rides, safari tram buses and more. The park changed name to Jungleland USA in 1956 after Disneyland was established. The park later went bankrupt in May 1969, due to competition from parks such as Disneyland, Knott's Berry Farm and Universal Studios. The park's 1,800 animals were sold at a public auction in October 1969.

The City of Thousand Oaks was incorporated on October 7, 1964, the first incorporated city in the Conejo Valley. Some sources mistakenly state that Thousand Oaks was incorporated on September 29, 1964, which was the date that voters approved the incorporation and selected the name. However, the incorporation only became official once the certificates of election were filed with the California Secretary of State, and then the record of affidavit was filed with the Ventura County Clerk.

The results of the cityhood election was clear on September 24, 1964. 2,780 residents voted to set up a city, while 1,821 had voted no to incorporation. Certain areas however tried to set up its own municipality. An attempt at a cityhood election in Newbury Park failed in 1963, as Talley Corporation and Janss Rancho Conejo Industrial Park refused to join the efforts. Reba Hays Jeffries, a local opponent of cityhood, told interviewers why she thought the cityhood election failed: Cityhood backers had to collect signatures from owners who represented 29% of the land that was to be incorporated. As the efforts collected 29% of registered voters, the measure never came on the ballot. Most Newbury Park land were annexed through the late 1960s and throughout the 1970s. Casa Conejo and Ventu Park are the only parts of Newbury Park left, which are not parts of Thousand Oaks. Lynn Ranch also decided to remain outside city limits.

Two-thirds of Westlake Village were annexed by Thousand Oaks in two portions - in 1968 and 1972. The Westlake neighborhood of North Ranch remained an unincorporated area until January 1973, when Thousand Oaks approved the annexation of North Ranch. North Ranch borders Oak Park, an unincorporated area where voters have chosen not to be annexed into Thousand Oaks.

Thousand Oaks is encouraging mixed-use retail and housing development along the downtown portion of Thousand Oaks Boulevard. The city is "built-out" within the confines of the Conejo Valley and has adopted a smart growth strategy as there is no room for the sprawling suburban growth the city is known for.

Increased development in Moorpark and Simi Valley in the late 1990s and early 2000s caused the Moorpark Freeway (Highway 23) to become heavily congested during both morning and afternoon rush hours. A major widening project began in 2008 to alleviate most of this congestion. Because of its desirable environment and location, property values appreciated more than 250% in less than ten years, primarily during the mid-1990s to early 2000s.^{[citation needed]}

On March 30, 2016, California Lutheran University and the NFL Rams team reached an agreement that allowed the team to have regular season training operations at CLU's campus in Thousand Oaks for the next two years. The Rams paid for two practice fields, paved parking, and modular buildings constructed on the northwestern corner of the campus.

On November 7, 2018, a lone gunman killed 12 people in a mass shooting at the Borderline Bar & Grill. Just days later, as a vigil was held for those impacted by the shooting, the massive Hill Fire and Woolsey Fire threatened the community, burning homes across Ventura and Los Angeles Counties. The fires would continue through almost the entire month of November charring almost 100,000 acres and consuming multiple homes in the region as it burned through Agoura Hills, Westlake Village, and Thousand Oaks, all the way to the Malibu coastline.^{[citation needed]}

In February 2019, in what was called a response to the shooting, members of the Westboro Baptist Church demonstrated outside Thousand Oaks High School. Parents, students, faith communities, and community members held a counter-demonstration across the street.

The city neighborhoods were built for the blue- and white-collar class in the 1950s. Today it is an upscale city with highly educated residents. The 2010 United States Census reported that Thousand Oaks had a population of 126,683. The population density was 2,295.8 people per square mile (886.4/km²). The racial makeup of Thousand Oaks was 101,702 (80.3%) White, 1,674 (1.3%) African American, 497 (0.4%) Native American, 11,043 (8.7%) Asian, 146 (0.1%) Pacific Islander, 6,869 (5.4%) from other races, and 4,752 (3.8%) from two or more races. Hispanic or Latino of any race were 21,341 persons (16.8%). The largest ancestry group is German-Americans 20,381 (15.8%), followed by Mexican 16,640 (12.9%), English 15,092 (11.7%), Irish 13,802 (10.7%), Italian 9,287 (7.2%), Russian 4,385 (3.4%), Chinese 4,256 (3.3%), French 4,127 (3.2%), Polish 4,127 (3.2%), Scottish 3,482 (2.7%), Indian 3,482 (2.7%), Norwegian 2,837 (2.2%) and Swedish 2,579 (2%).

The census reported that 124,941 people (98.6% of the population) lived in households, 1,390 (1.1%) lived in non-institutionalized group quarters, and 352 (0.3%) were institutionalized.

There were 45,836 households, out of which 16,439 (35.9%) had children under the age of 18 living in them, 27,206 (59.4%) were opposite-sex married couples living together, 4,260 (9.3%) had a female householder with no husband present, 1,925 (4.2%) had a male householder with no wife present. There were 1,761 (3.8%) unmarried opposite-sex partnerships, and 284 (0.6%) same-sex married couples or partnerships. 9,728 households (21.2%) were made up of individuals, and 4,459 (9.7%) had someone living alone who was 65 years of age or older. The average household size was 2.73. There were 33,391 families (72.8% of all households); the average family size was 3.15.

The population was spread out, with 30,076 people (23.7%) under the age of 18, 10,226 people (8.1%) aged 18 to 24, 29,853 people (23.6%) aged 25 to 44, 37,964 people (30.0%) aged 45 to 64, and 18,564 people (14.7%) who were 65 years of age or older. The median age was 41.5 years. For every 100 females, there were 95.8 males. For every 100 females age 18 and over, there were 93.3 males.

There were 47,497 housing units at an average density of 860.8 per square mile (332.3/km²), of which 33,501 (73.1%) were owner-occupied, and 12,335 (26.9%) were occupied by renters. The homeowner vacancy rate was 0.8%; the rental vacancy rate was 5.6%. 92,510 people (73.0% of the population) lived in owner-occupied housing units and 32,431 people (25.6%) lived in rental housing units.

The median income for a household in the city was $121,088.