Hawking looks set to star as himself in his motorised wheelchair, replacing Matt Lucas’ character Andy.
It is not thought Lucas will take part in the sketch, but it has yet to be confirmed whether he will appear or not.
In January, Walliams was seen filming the Comic Relief sketch with Catherine Tate at St John's College, Cambridge, who was dressed as a nun for the skit.
The award-winning comedy Little Britain ran for four series between 2003 and 2006. Walliams last reprised his role as Lou for the Pride of Britain Awards in 2009.
I just finished an extraordinary two days in Cambridge shooting a sketch for @rednoseday with Catherine Tate & Professor Stephen Hawking.— David Walliams (@davidwalliams) January 28, 2015
Comic Relief airs on BBC1 on Friday 13 March and will be broadcast from the London Palladium for the first time.
Claudia Winkleman, Davina McCall and Lenny Henry are presenting, while Dawn French, Mr Bean and Dermot O’Leary are also on board for the comedy show.