The 54-year-old had needed to run a double marathon on Sunday, finishing beneath a giant statue of Nelson Mandela in Johannesburg.
Izzard attempted a similar feat in South Africa in 2012 but had to pull out for health reasons.
It has been confirmed the comedian's endurance feat had raised more than Â£1.35m for the charity appeal.
"That was very, very tough," he told reporters as he swigged from a celebratory bottle of sparkling white wine.
The 27 marathons were intended to reflect the 27 years Mr Mandela had spent in prison before becoming South Africa's first black president.
"It's been the hardest thing I've ever done," said a "very tired" Izzard. "Thank you to everyone who has donated [and] don't do this at home."
Gary Lineker, marathon runner Paula Radcliffe and DJ Chris Moyles are among those who have tweeted their congratulations.
Comedian Eddie Izzard has completed his challenge in South Africa to run 27 marathons in 27 days for Sport Relief.