Janssen was born in 1931 in Naponee, a village in Franklin County in southern Nebraska, to Harold Edward Meyer, a banker (May 12, 1906 – November 4, 1990) and Berniece Graf (May 11, 1910 – November 26, 1995). Janssen was of Irish and Jewish descent. Following his parents' divorce in 1935, his mother moved with five-year-old David to Los Angeles, California, and later married Eugene Janssen (February 18, 1918 – March 30, 1996) in 1940 in Los Angeles. Young David used his stepfather's name after he entered show Business as a child.

Janssen appeared in many television series before he landed programs of his own. In 1956, he and Peter Breck appeared in John Bromfield's syndicated series Sheriff of Cochise in the episode "The Turkey Farmers". Later, he guest-starred on NBC's medical drama The Eleventh Hour in the role of Hal Kincaid in the 1962 episode "Make Me a Place", with series co-stars Wendell Corey and Jack Ging. He joined friend Martin Milner in a 1962 episode of Route 66 as the character Kamo in the episode "One Tiger to a Hill."

Janssen was married twice. His first marriage was to model and interior decorator Ellie Graham, whom he married in Las Vegas on August 25, 1958. They divorced in 1968. In 1975, he married Actress and model Dani Crayne Greco. They remained married until Janssen's death.

He starred as a Los Angeles police detective trying to clear himself in the killing of an apparently innocent Doctor in the 1967 film Warning Shot. The film was shot during a break in the spring and summer of 1966 between the third and fourth seasons of The Fugitive.

Janssen played an alcoholic in the 1977 TV movie A Sensitive, Passionate Man, which co-starred Angie Dickinson, and an Engineer who devises an unbeatable system for blackjack in the 1978 made-for-TV movie Nowhere to Run, co-starring Stefanie Powers and Linda Evans. Janssen's impressively husky voice was used to good effect as the narrator for the TV mini-series Centennial (1978–79); he also appeared in the final episode. He starred in the made for tv mini series “S.O.S. Titanic” as John Jacob Astor, playing opposite Beverly Ross as his wife, Madeleine, in 1979.

A heavy drinker and a four-pack-a-day smoker, Janssen died of a heart attack in the early morning of February 13, 1980, at his home in Malibu, California at the age of 48. At the time of his death, Janssen was filming the television movie Father Damien. Janssen was buried at the Hillside Memorial Park Cemetery in Culver City, California. A non-denominational funeral was held at the Jewish chapel of the cemetery on February 17. Suzanne Pleshette delivered the eulogy at the request of Janssen's widow. Milton Berle, Johnny Carson, Tommy Gallagher, Richard Harris, Stan Herman, Rod Stewart and Gregory Peck were among Janssen's pallbearers. Honorary pallbearers included Jack Lemmon, George Peppard, James Stewart and Danny Thomas.

In 1996 TV Guide ranked him number 36 on its 50 Greatest TV Stars of All Time list.