O'Keefe started in films as an extra in the early 1930s and appeared in numerous films under the name Bud Flanagan. After a small but impressive role in Saratoga (1937), Clark Gable recommended O'Keefe to Metro-Goldwyn-Mayer, which signed him to a contract in 1937 and renamed him Dennis O'Keefe. His film roles were bigger after that, starting with The Bad Man of Brimstone (1938), and the lead role in Burn 'Em Up O'Connor (1939).
O'Keefe left MGM around 1940 but continued to work in mostly lower budget productions. He often played the tough guy in action and crime dramas but was also known as a comic actor as well as a dramatic lead.
He gained great attention with a showy role in The Story of Dr Wassell and became a comedy star. He expressed interest in expanding into direction.
In the 1950s he did some directing and wrote mystery stories. In the middle 1950s, he appeared on NBC's legal drama Justice and on the network's The Martha Raye Show. On October 3, 1957, he was a guest star on another NBC variety show, The Ford Show, starring Tennessee Ernie Ford. From 1957 to 1958, he was the host of Suspicion, a TV series produced by Alfred Hitchcock. From 1959-1960, he was the star of the CBS Television situation comedy, The Dennis O'Keefe Show.
A heavy cigarette smoker, O'Keefe died of lung cancer in 1968 at the age of sixty. Ironically, in his film The Diamond Wizard (1954), he directed and stars in, he played a U.S. Treasury Agent in London. In the film, O'Keefe's character is trying to quit smoking. Throughout the film, he is prevented from lighting up by different characters in the film. After the rousing finale, however, as a reward for his heroism, he is finally allowed to light up. He takes a deep puff and joyously exhales.