Rooney missed United's 1-0 victory at Wolverhampton Wanderers with a knee injury which he aggravated playing the full 90 minutes for England against Egypt mid-week.
"Rooney is very doubtful for the Milan game unfortunately," Ferguson told MUTV. "It's one of these bad bits of luck you get - the Wembley pitch has just killed him."
Ferguson had criticised his leading striker for playing from start to finish in England's friendly on a leg-sapping Wembley pitch, just three days after scoring the winner in the League Cup final.
"It is his own enthusiasm that has caused it. His problem is that he can't say no," Ferguson said of Rooney who has scored 23 Premier League goals this season.
United won the opening leg of their first knockout round tie 3-2 in the San Siro but the loss of Rooney for Wednesday's match would be a blow, especially with Michael Owen ruled out for the season.
Ferguson said Dimitar Berbatov would most likely start against Milan.
"I was pleased with how Berbatov did against Wolves though and I'll probably start with him on Wednesday," Ferguson said.
