Before becoming an actor he was a local Artist in Seattle and, later, Hollywood, working on several of the murals inside Grauman's Chinese Theatre. He did some of the original artwork for the 1933 King Kong pressbook. Luke also painted the casino's mural in The Shanghai Gesture. He published a limited edition set of pen and ink drawings of The Rubiyat of Omar Khayyam in the 1950s. He also created illustrations for the books The Unfinished Song of Achmed Mohammed by Earle Liederman, Blessed Mother Goose by Frank Scully and an edition of Messer Marco Polo by Brian Oswald Donn-Byrne (unpublished). Other art done by Luke included the dust jackets for books published in the 1950s and 1960s. It was through his studio art work that he was recruited for his first movie roles.

Luke made his film debut in The Painted Veil (1934), and the following year gained his first big role, as Charlie Chan's eldest son in Charlie Chan in Paris. He worked so well with Warner Oland, the actor playing Chan, that "Number One Son" became a regular character in the series, alternately helping and distracting 'Pop' Chan in each of his murder cases. Luke appeared seven times as Lee Chan opposite Oland's Chan. Keye Luke left the Charlie Chan series in 1938, shortly after Oland died. The unfinished Oland-Luke film Charlie Chan at the Ringside was completed as Mr. Moto's Gamble, with Luke now opposite Peter Lorre.

Luke was born in Guangzhou, China, to a father who owned an art shop, but grew up in Seattle. He was part of the Luke family, a relative of Wing Luke, for whom Seattle's Wing Luke Asian Museum was named. He had four siblings who all emigrated from China to California during the Depression. His younger brother Edwin Luke also became an actor in the Charlie Chan series. In Seattle, Luke attended Franklin High School, where he contributed cartoons and illustrations to school publications. Keye Luke became a naturalized citizen of the United States in 1944—in a moment fictionally recreated in Lisa See's novel Shanghai Girls.

RKO Radio Pictures used Luke in its popular adventures of The Falcon and Mexican Spitfire. Luke also worked at Universal Pictures, where he played two-fisted valet/chauffeur Kato in its Green Hornet serials. In 1946 Universal mounted a low-budget serial consisting largely of action footage from older films; Keye Luke was hired to match old footage of Sabu in the serial Lost City of the Jungle.

In 1948, Keye Luke returned to the Chan mysteries, which were now being produced by Monogram and starred Roland Winters as Chan. "Number One Son" appeared in the last two Chan features, The Feathered Serpent along with "Number Two Son" Tommy Chan (Victor Sen Yung) in their only appearance together, and Sky Dragon. In both of these films, Luke was older than the actor playing his father.

In 1958, Luke had a featured Broadway role in the Rodgers and Hammerstein musical Flower Drum Song, directed by Gene Kelly. The Soundtrack album captures his singing of the part of Mr. Wang, the family patriarch.

Luke played Governor Donald Cory in a 1969 episode of Star Trek entitled "Whom Gods Destroy", and was going to play Doctor Noonien Soong in the Star Trek: The Next Generation episode "Brothers" but illness prevented him from doing so; Brent Spiner ultimately took over the role.

Keye Luke also worked extensively in television, making numerous guest appearances, including four on The F.B.I. and seven TV movies. He was a regular cast member in two short lived sitcoms, Anna and the King starring Yul Brynner in 1972 and Sidekicks in 1986-87. Also in 1972, "Number One Son" ascended to the role of Charlie Chan himself, thus becoming the first actor of Chinese descent to play the role: he supplied the voice of "Mr. Chan" in the animated television series The Amazing Chan and the Chan Clan. He was also known for his role of Master Po in the television series Kung Fu (1972–75). In 1985, Luke played 'The Ancient One' on the soap opera General Hospital, for the Asian Quarter storyline, which showcased strong chemistry between Luke and young Actress Kimberly McCullough, whom he mentored. In 1986, Luke appeared in season two of "The Golden Girls" as Sophia's love interest. Additionally Luke voiced many animated series including Brak in Space Ghost, the aforementioned Charlie Chan, and Zoltar/The Great Spirit/Colonel Cronus in Battle of the Planets.

Luke died of a stroke on January 12, 1991. He was 86 years old and is buried at Rose Hills Memorial Park in Whittier, California.

Writer and filmmaker Timothy Tau wrote, directed and produced a short film about Keye Luke's earlier life and work, entitled Keye Luke, which premiered at the 2012 Los Angeles Asian Pacific Film Festival as a Visual Communications "Armed with a Camera" Fellowship film. The film was also Closing Night Film of the inaugural 2013 Seattle Asian American Film Festival. Feodor Chin starred as Keye Luke. Archie Kao starred as Edwin Luke, Keye Luke's brother. Kelvin Han Yee starred as Lee Luke, Keye Luke's father.