The Italian football federation (FIGC) confirmed that Marchisio underwent tests which have revealed serious damage to his right knee and is expected to be sidelined for six months.
A statement from the FIGC read: “During (Friday’s) warm-up, Marchisio sustained trauma in his right knee.
“An MRI scan was immediately carried out in Florence and it has revealed a subtotal lesion of the anterior cruciate ligament.
“After informing the Juventus medical staff, the player will return to his club.”
His injury is a big blow for Italy and for the Bianconeri, who are still alive in three competitions this season.
Juve are 14 points clear of Roma at the top of Serie A with 10 games remaining.
Massimiliano Allegri’s side are into the quarter-finals of the Champions League and the semi-finals of the Coppa Italia.
The Turin giants take on Ligue 1 high-flyers Monaco next in Europe’s elite club competition.
The 29-year-old has scored two goals and set up five more in 27 league appearances for the Serie A leaders.
Italy take on Bulgaria in Sofia in Saturday’s Euro 2016 qualifier before hosting England in an international friendly in Turin three days later.