The Australian pair performed a duet of their 1988 hit Especially For You during the charity marathon on BBC One.
A fundraising grand total of £51,242,186 was announced at the end of the broadcast, beating the record £50.4m raised during the last Sport Relief show two years ago.
The charity marathon was hosted live from the Queen Elizabeth Olympic Park in east London by presenters Gary Lineker and David Walliams. A host of stars featured in sketches to get the nation active and persuade them to donate to good causes that help thousands of people in the UK and across the world.
Highlights included David Beckham starring in the first new episode of Only Fools And Horses for over 11 years. The show also included the first airing of the England football team’s World Cup song, a cover version of Take That’s Greatest Day.
Gary Barlow and former England captain Lineker led a 20-strong group including pop stars Katy B, former Spice Girl Emma Bunton, Pixie Lott and Conor Maynard.
Footballers Peter Shilton, Michael Owen, Glenn Hoddle, Dion Dublin and Sir Geoff Hurst also took part.
All proceeds from the sale of the record will go to Sport Relief.
An Olympic-themed show also featured gold medallist Lord Coe taking part in a Clash of the Titans battle against comedian John Bishop at the London velodrome.