The Spain international only returned to training on Thursday after sustaining a broken hand against Valencia on January 23 and, although he travelled with the squad, he will not be considered.
"Everyone is fit and ready to play and be considered, with the exception of Iker Casillas," Mourinho said ahead of the second leg of the last-16 tie.
"We are sorry we had to travel yesterday (because of a baggage handlers strike) because we don't like to wait.
"The emotion is high and we want to play. Tomorrow the world will stop to watch this tie, which doesn't look like a tie it looks like a final."
The match marks the first return of ex-United forward Cristiano Ronaldo to Old Trafford, and much of the pre-match focus has been on him with Sir Alex Ferguson having spoken in glowing terms about his former charge.
"I think he is fine, as far as I am aware," the Madrid boss added.
"I think any accolade he gets is more than deserved and I think the reception he will get from the crowd will be deserved. I am aware how well he did here and what an impact he had at the club."