The 26-year-old has been in good form this season after finally getting over knee injuries in recent campaigns, and his performances in Serie A had earned him a place in Prandelli's squad.
Rossi has scored 11 goals in 12 league matches this season to help propel Fiorentina into fifth place in the league, and make him the leading goalscorer in the division.
However, a bout of tonsillitis has laid him low and national boss Prandelli confirmed he was not fit enough to play despite taking part in training ahead of the match in Milan on Friday.
"Rossi definitely won't be there, even though he took part in the first training session today (Thursday)," Prandelli said. "(Daniele) De Rossi is available but will most likely start on the bench."
Prandelli also named Southampton striker Pablo Daniel Osvaldo as Rossi's replacement and the 27-year-old will partner Mario Balotelli – who the head coach believes is looking better than ever.
"I've never seen him (Balotelli) so well and so motivated," he added. "A great player knows that he is always under scrutiny and that each match hides pitfalls.
"He is a player who is feared all the world over."
Goalkeeper Gianluigi Buffon will make his 138th appearance for Italy in the match but may be rested for their fixture against Nigeria on Monday.
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