The comedian and talkshow host celebrated the milestone during last night's broadcast of the long-running late-night television show.
Leno took over the role as host after TV fixture Johnny Carson stepped down from the position in 1992.
The host was plunged into controversy in 2009, after he handed down his post as host of The Tonight Show to Conan O'Brien, only to return to the position in March 2010.
During his opening monologue on NBC, the 62-year-old Leno said: "Today is our anniversary. 20 years ago today, we did our very first show. In that time we've seen a lot of stars come and go from the programme… including me!"
Guests during the anniversary celebration included Jack Black, Meatball Shop Guys and Colin Powell. Musician Beirut also performed.
Watch Jay Leno's opening monologue below: