De Lancie was born John Sherwood de Lancie, Jr. in Philadelphia, Pennsylvania on November 13, 1948, one of two children born to John Sherwood de Lancie, Sr. (1921–2002), principal oboist of the Philadelphia Orchestra from 1954 to 1977, and Andrea de Lancie (d. 2006). He has a sister named Christina (born November 21, 1953).

On March 20, 1984, de Lancie married actress Marnie Mosiman (born March 6, 1951), who guest-starred as Harmony / Wisdom / Balance in “Loud as a Whisper,” an episode of Star Trek: The Next Generation. The couple have two sons: Keegan (born 1984) and Owen (born 1987).

De Lancie co-wrote the Star Trek novel I, Q with Peter David, as well as co-writing the novel Soldier of Light (with Tom Cool). He wrote the DC comic book story The Gift. In 1996 with Leonard Nimoy and writer-producer Nat Segaloff, De Lancie formed and recorded Alien Voices, a collection of audio dramas based on classic science fiction and fantasy stories, such as The Time Machine and The Lost World.

De Lancie co-hosted and appeared in Star Trek: The Music, a touring company, with fellow actor Robert Picardo, who portrayed the Doctor in Star Trek: Voyager. De Lancie and Picardo narrate around the orchestral performance, explaining the history of the music in Star Trek. He performed Pierre Curie in Alan Alda’s play, Radiance: The Passion of Marie Curie in 2001 at the Geffen Theater in Los Angeles.

De Lancie was the writer, director and host of First Nights, an adult concert series at the Walt Disney Concert Hall with the Los Angeles Philharmonic, based loosely on the book of the same name by Thomas Forrest Kelly, which explored the life and music of Stravinsky, Beethoven, Mahler, Schumann, and Prokofiev. In 2006, de Lancie made his opera directorial debut with the Atlanta Opera performing Puccini’s “Tosca” from May 18–21.

In a 2012 interview, de Lancie recalled his original audition for Q, after which Roddenberry approached him and said, “You make my writing sound better than it is.”

He started a Kickstarter campaign to help fund the documentary, now titled Bronies: The Extremely Unexpected Adult Fans of My Little Pony. The Kickstarter campaign began on May 13, 2012 and by June 10, had reached a total of $322,022, becoming Kickstarter’s second-highest funded film project of all time.

On June 4, 2016, he addressed the participants at the Reason Rally in Washington, D.C. Speaking in reference to his Star Trek character Q, he said:

On July 14, 2017, he attended the unveiling of a statue of Clarence Darrow at the Rhea County Courthouse, Dayton, Tennessee, the site of the Scopes Trial in 1925, where Darrow had argued in favor of the teaching of evolution and secular education.

In September 2019, de Lancie narrated as “Forever of the Stars” in the first ever live performances of Ayreon’s Sci-Fi concept album Into the Electric Castle.

In October 2019, de Lancie was a featured speaker at the annual conference of the Center for Inquiry, CSICon. At the conference he announced two new projects. The first, an animated series, titled God’s Goofs, is meant to point out that intelligent design is absurd. The second project is a play based on the 2005 intelligent design trial in Dover, Pennsylvania.