Ambrosini made his international debut in April 1999 against Croatia and was part of the Italian squad at the 2000 Summer Olympics and at Euro 2000, which ended with an appearance as a substitute in the final loss to France. Ambrosini was unavailable for selection for the 2002 FIFA World Cup finals through injury and then failed to break into the squad for the Euro 2004 finals.
On 16 August 2006, Ambrosini earned his 23rd cap for Italy after almost 2 years of absence from the Italian squad. For this match, he was given the captain's armband by coach Roberto Donadoni in the absence of regular contenders for the armband.
