Ambrosini, who spent almost 20 years at Milan before he was shown the door after last season, was expected to miss the clash but has recovered to make the Viola's matchday squad.
The 36-year-old says he will enjoy going back to the San Siro with his new team but believes Milan have no regrets about letting him depart.
"I don't think they miss me," Ambrosini told the Gazzetta dello Sport.
"Nigel De Jong is always the best on the pitch, with Ricardo Montolivo or Sulley Muntari.
"I don't see why they would have any regrets. They are behind (Fiorentina) in the standings but they remain a great club and a great squad.
"Joining Fiorentina was a challenge for me. There are few clubs in Italy with real ideas and the Viola are one of them."
While Ambrosini has made the cut, David Pizarro (leg), Marko Bakic (ankle) and Mati Fernandez are all out through injury.
Juan Cuadrado is also unavailable due to a one-match ban.
Despite the absence of a host of key players, Fiorentina boss Vincenzo Montella expects his side to bounce back after their midweek defeat to Napoli, which has dropped them back to sixth spot on the Serie A ladder.
"Nobody said that getting into the Champions League should be our goal," Montella said.
"We just want to always give our best and try to entertain people – for me that is the most important thing.
"But everyone recognises that we have to improve at the back and that starts from the front.
"We have to react after a defeat but football is like that. Sometimes the game is unfair, but you have to get on with it."
Milan have injury problems of their own with young defender Mattia De Sciglio ruled out after picking up a knock during training this week.
