John Cleese, Michael Palin, Eric Idle, Terry Gilliam and Terry Jones performed together for the first time in decades at London's O2 Arena on Tuesday night.
The show, which featured an extended cast of dancers, a full orchestra and special effects, ended with a singalong version of Always Look On The Bright Side of Life.
Among the classic sketches was the Four Yorkshiremen, comparing their tough upbringing, and the Lumberjack Song.
The audience was also treated to clips from old shows including the fish-slapping sketch, and some of the biggest cheers of the night were reserved for archive clips featuring sixth Python Graham Chapman who died in 1989.
The second half of the show kicked off with a spoof ballet - Spam Lake - before a performance of the X-rated Sit On My Face song. Other sketches included Idle and Palin as lingerie-wearing judges and Idle and Jones performing the "nudge, nudge, wink, wink" routine which inspired a song and dance number.
Fans applauded every aspect of the show which included big song and dance routines which allowed time for the numerous costume changes.
Stephen Fry turned up on stage later in the night during a sketch about a game show host blackmailing misbehaving celebrities, and other famous faces included Professor Brian Cox and Stephen Hawking, who appeared on film after a performance of The Galaxy Song.
Monty Python's Flying Circus was made for TV between 1969 and 1974 and generations of fans can recite lines and whole sketches.
The last ever gig on 20 July will be broadcast in cinemas around the world.