Roma, second in Serie A, said in a statement that the 25-year-old had ruptured the anterior cruciate ligament during the second half of Wednesday's 3-1 Serie A win at Sassuolo.
The Italy international will undergo surgery later on Thursday, the club said.
Florenzi has won 24 caps for Italy and started in four of their five games at Euro 2016 when they reached the quarter-finals.
He played in the first two of their qualifiers for the 2018 World Cup, although he was left on the bench for the most recent game in Macedonia.
(Writing by Brian Homewood, editing by Nick Mulvenney)
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