Brazil-born Napoli midfielder Jorginho and Fiorentina striker Federico Bernardeschi have both been called up for the first time. Thiago Motta has also been included after impressing for Paris Saint-Germain this season.
However Balotelli, who has scored 13 goals in 33 appearances for the Azzurri, has not been selected.
The 25-year-old has struggled with injuries and poor form at AC Milan this season on loan from Liverpool, and his omission from Conte's squad will raise doubts over the striker's chances of playing at Euro 2016 this summer.
Antonio Conte is expected to leave his role as Italy coach at the end of Euro 2016
Conte has recently signaled his intention to quit his role with Italy after the upcoming tournament in France - further fueling speculation about a potential move to Stamford Bridge.
Italian federation president Carlo Tavecchio confirmed Conte's plans to move into club football later this year.
"Antonio Conte has told me that at the end of the European championship, his job will end," he said. "He feels the need for the pitch, the everyday training, and this is something you can understand."
