Centre-back Bonucci, who recently joined Union Berlin, did not make the cut despite being Italy's most recent captain.
Locatelli played an instrumental role in Italy’s Euro 2020 triumph but had not been called up since 2022 by previous manager Roberto Mancini.
He will be joined by other long-term absentees Mattia Zaccagni of Lazio and AS Roma's Gianluca Mancini, while Lazio defender Nicolo Casale received his first call-up.
Spalletti's debut in charge will be Italy's Euro 2024 qualifier in North Macedonia on September 9th before the Azzurri host Ukraine three days later.
Spalletti led Napoli last season to their first Serie A title in 33 years and took over the national team after Mancini resigned last month.
Italy are third in Group C on three points from two matches, trailing leaders England who have 12 from four and Ukraine on six points from three matches.
Squad:
Goalkeepers: Gianluigi Donnarumma (PSG), Alex Meret (Napoli), Ivan Provedel (Lazio), Guglielmo Vicario (Tottenham Hotspur)
Defenders: Alessandro Bastoni (Inter Milan), Cristiano Biraghi (Fiorentina), Nicolo Casale (Lazio), Matteo Darmian (Inter), Giovanni Di Lorenzo (Napoli), Federico Dimarco (Inter), Gianluca Mancini (AS Roma), Alessio Romagnoli (Lazio), Giorgio Scalvini (Atalanta), Leonardo Spinazzola (Roma)
Midfielders: Nicolo Barella (Inter), Bryan Cristante (Roma), Davide Frattesi (Inter), Manuel Locatelli (Juventus), Lorenzo Pellegrini (Roma), Matteo Pessina (Monza), Sandro Tonali (Newcastle United)
Forwards: Federico Chiesa (Juventus), Wilfried Gnonto (Leeds United), Ciro Immobile (Lazio), Matteo Politano (Napoli), Giacomo Raspadori (Napoli), Mateo Retegui (Genoa), Mattia Zaccagni (Lazio), Nicolo Zaniolo (Aston Villa)
