Walsh was set to ride the Mullins-trained Sir Des Champs in Saturday's race, but the 36-year-old Irishman injured his wrist in two falls at Aintree on Friday.
"Unfortunately, Ruby does not ride tomorrow," Mullins said on Twitter, having earlier played down the severity of the jockey's injury.
Walsh won the Grand National at his first attempt on Papillon in 2000 and again in 2005 on Hedgehunter.
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Nina Carberry will stand in for Walsh on Sir Des Champs.
(Reporting by Toby Davis; editing by Ken Ferris)
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