Fiorentina said the extent of the damage would be known on Monday and coach Vincenzo Montella is worried about Rossi, who was taken off after a rough challenge by Livorno defender Leandro Rinaudo in the 71st minute.
Rossi, who has come storming back this season after recovering from successive anterior cruciate ligament injuries which effectively cost him two years of his career, limped away in pain and looking worried.
Rinaudo was booked and was lucky not to be sent off after he argued with the referee and his opponents, leading to a series of pushes among the players.
Fiorentina had taken the lead five minutes earlier when defender Gonzalo Rodriguez headed in from a corner.
"We are all worried," coach Vicenzo Montella told reporters. "It appeared to be an intentional foul. I don't think he wanted to injure the player but he certainly wanted to stop him.
"I expected that Rinaudo would have apologised which you usually do when you commit that sort of foul," he added.
The club issued a statement confirming an injury to Rossi's right knee.
"Fiorentina announce that following the challenge by the Livorno player Rinaudo, Giuseppe Rossi has suffered a sprain to the knee which has previously been operated on.
"More tests will be carried out tomorrow."
The American-born striker's troubles began when he suffered an anterior cruciate ligament injury while playing for Villarreal in a Spanish league match against Real Madrid in 2011.
He fought desperately to be fit again in time Euro 2012 only to re-injure the knee in training the following April, causing him to miss the tournament.
Villarreal sold him to Fiorentina at the start of this year and he finally made his comeback against Pescara in May, playing 27 minutes in the final match of last season.
His performances this season, with 14 Serie A goals, have also earned him an Italy recall.
"Rinaudo didn't apologise and even argued with the referee in an undignified manner," said Fiorentina owner Andrea Della Valle. "He should look at the replays and reflect on his behaviour."
The injury overshadowed the win which lifted Fiorentina to joint third alongside Napoli, who host Sampdoria on Monday, on 36 points from 18 games.
Earlier, Cagliari forward Mauricio Pinilla missed a penalty, sending a wild kick high and wide of the goal, as his side drew 0-0 at Chievo in a match which kicked off the weekend programme.