Charles shares a joke with Will Smith and Michael Mann
The film's star greeted the hundreds of screaming fans who braved the cold of London's Leicester Square on Tuesday, and signed autographs before the screening.
It was hoped Ali, who suffers from Parkinson's Disease, would be a special guest but he cancelled his appearance because of ill health.
Prince Charles instead met heavyweight boxing champion Lennox Lewis and former British title holder Henry Cooper, who hosted the gala event.
The boxers were joined by Olympic gold medallist Audley Harrison and former champions Henry Cooper and Barry McGuigan.
Prince Charles arrived a few minutes before the movie was due to start and met staff at the Empire Cinema, top executives involved in the film and its director Michael Mann, whose previous work has included Manhunter, Heat and the critically-acclaimed The Insider.
The Prince also met Smith, who told him he had spent around six hours a day sparring and training for the role.
Comedians Frank Skinner and Harry Enfield, Arsenal's Patrick Vieira and former Gunners' striker Ian Wright, veteran singer Bob Geldof, Skunk Anansie lead singer Skin, BBC Radio 1 DJ Sara Cox, Tamara Beckwith and Lady Victoria Hervey also attended.
The movie Ali is already being tipped as an Oscar contender.
Men in Black star Smith put on two stone (12.7 kg) to play the boxing hero over a 10-year period, covering his belt-winning 1964 fight with Sonny Liston and his conversion to Islam.
Ali gave his blessing to the production and was reportedly pleased with the casting of Smith in the central role.
The movie cost £73m to make, making it the most expensive biopic ever produced.
But there were doubts the film would be completed after the studio grew concerned at escalating costs.
But after discussions Mann agreed to pay for any costs that ran over budget, reduce fees, lower his share of foreign profits and cuts scenes from the film.
Other stars of the film include Jamie Foxx, John Voight, Mario van Peebles and Michael Michele of ER fame.
Smith's wife Jada Pinkett-Smith plays Ali's first wife Sonji Roi and former WBO heavyweight champion Michael Bentt plays Liston.
The royal première was held in aid of the Cinema and Television Benevolent Fund.
It premières in the US on Wednesday and opens later in December.
But the film does not open in the UK until February.