The soap foursome - better known as Carl King, John Barton, Andy Sugden and Nikhil Sharma - celebrated on Twitter after successfully riding from the ITV show's set to EastEnders' iconic Queen Vic pub in little over 20 hours.
The group, who were joined by executive producer Steve November and a gang of professional cyclists, have so far raised for Macmillan almost £43,000 of their £100,000 target.
"We have only gone and bloody DONE IT," Thornton wrote. "224 miles in under 20.5 hours!!!! Get in! that was EPIC! #teamemmerdale."
Fletcher also exclaimed on the social networking site: "Finished it!!!!!!!! Get in! Amazing! What a journey. Some grim times and some great laughs. Very proud! We smashed it!"
The mammoth challenge was set up in memory of late Emmerdale producer Gavin Blyth, who passed away after a short battle with cancer last November.
Donations for the Emmerdale to EastEnders challenge can be made via a dedicated JustGiving page. Alternatively, a £5 donation can be made by texting 'RIDE66 £5' to 70070.