Barella, a key figure in Spalletti's plans, sat out the 0-0 draw with Turkey on Tuesday, but his minor muscle strain is not enough to keep the Inter Milan man out of the squad.
Federico Gatti, who was added to the provisional squad after the withdrawal of defenders Francesco Acerbi and Giorgio Scalvini, keeps his place in the group for Germany, while Ivan Provedel is the keeper who misses out.
Provodel has been told to continue training, as there are doubts over the fitness of Napoli keeper Alex Meret.
Samuele Ricci and Riccardo Orsolini are the other two players from the provisional squad to miss out. Orsolini started the scoreless draw with Turkey, but the Bologna forward did not do enough to earn a place in the final squad.
His Bologna teammate Riccardo Calafiori came on as a late substitute in that game to make his international debut, and the defender is included in Spalletti's list.
Juventus midfielder Nicolo Fagioli, who recently returned from a seven-month ban for breaching rules on betting, is also included, having come off the bench against Turkey for his second cap.
Italy have one more warm-up game against Bosnia on Sunday, before they head to Germany to defend their title, in a tough Group B where they will face Spain, Croatia and Albania.
Italy squad for Euro 2024:
Goalkeepers: Gianluigi Donnarumma (Paris St Germain), Alex Meret (Napoli), Guglielmo Vicario (Tottenham Hotspur)
Defenders: Alessandro Bastoni (Inter), Raoul Bellanova (Torino), Alessandro Buongiorno (Torino), Riccardo Calafiori (Bologna), Andrea Cambiaso (Juventus), Matteo Darmian (Inter), Giovanni Di Lorenzo (Napoli), Federico Dimarco (Inter), Federico Gatti (Juventus), Gianluca Mancini (Roma)
Midfielders: Nicolo Barella (Inter), Bryan Cristante (Roma), Nicolo Fagioli (Juventus), Michael Folorunsho (Hellas Verona), Davide Frattesi (Inter), Jorginho (Arsenal), Lorenzo Pellegrini (Roma)
Forwards: Federico Chiesa (Juventus), Stephan El Shaarawy (Roma), Giacomo Raspadori (Napoli), Mateo Retegui (Genoa), Gianluca Scamacca (Atalanta), Mattia Zaccagni (Lazio)
