DeLuise made his film debut in the 1979 film Hot Stuff. He landed his best known acting role, as Officer Doug Penhall, in the 1987 Fox series 21 Jump Street, alongside other promising actors including Johnny Depp. His brother Michael came on the show in the fifth season where he played his younger brother, Officer Joey Penhall. DeLuise is also well known for his role as Dagwood on the NBC science fiction television series SeaQuest DSV from 1994 to 1996.
In 1997, he began working on the series Stargate SG-1, serving as Producer, Writer, Director, and creative consultant. He has appeared, normally as an extra, in every episode of the series he has directed. He went on to work as executive Producer, Director and Writer for the Stargate SG-1 spin-off Stargate Atlantis, and directed eight episodes of the spin-off Stargate Universe. His father, Dom DeLuise, made a guest appearance in the Stargate SG-1 episode "Urgo", which Peter directed. Peter also made a cameo in the episode as the "Urgo" character transformed to be a young man in a United States Air Force uniform.
DeLuise was previously married to Gina Nemo, they divorced in 1992. He later married Actress Anne Marie Loder on June 7, 2002; together they have a son, who was born on April 11, 2004.
DeLuise directed the CBC television series jPod, based on the novel of the same name by Douglas Coupland, which debuted in January 2008 and directed the fantasy film Beyond Sherwood Forest.
He made a brief cameo appearance alongside Johnny Depp in the 2012 film adaptation, 21 Jump Street. Depp, DeLuise and Holly Robinson reprised their roles as Tom Hanson, Doug Penhall and Judy Hoffs, respectively.