The Chelsea striker has struggled for fitness since the back end of last season, with the current campaign seeing a club-versus-country row develop between Stamford Bridge boss Jose Mourinho and Spain counterpart Vicente del Bosque.
Mourinho had suggested that Del Bosque overplayed Costa in the last round of internationals and the striker has not scored since returning to club action.
And Del Bosque has seemingly moved to avoid further squabbles by leaving Costa out of his latest selection, instead giving attacking pair Nolito and Jose Callejon their first call-ups.
"I would not have brought a player with health problems," said Del Bosque. "It has nothing to do with his club, but we have spoken and it is the best [solution] for his health."
Nolito has scored five goals for Celta Vigo this season and is the leading Spanish goalscorer in La Liga, while Callejon's return of eight strikes in 10 Serie A outings for Napoli is the best in Italy's top flight.
Malaga's Ignacio Camacho is handed a first senior call-up, while Alvaro Morata and Isco return to the fold with Andres Iniesta and David Silva missing due to injury.
Paco Alcacer, who has scored in all three Euro 2016 qualifiers Spain have played so far, retains his place, however there is no place for Jesus Navas of Manchester City.
Spain currently occupy second place in Group C behind Slovakia after three matches, a defeat in Zilina leaving them three points behind ahead of the next round of fixtures.
They face Belarus in qualifying on November 15, before welcoming Germany to Vigo for a friendly clash between the last two World Cup winners three days later.
Squad in full:
Goalkeepers: Kiko Casilla (Espanyol), Iker Casillas (Real Madrid), David de Gea (Manchester United)
Defenders: Jordi Alba (Barcelona), Raul Albiol (Napoli), Cesar Azpilicueta (Chelsea), Marc Bartra (Barcelona), Juan Bernat (Bayern Munich), Juanfran (Atletico Madrid), Gerard Pique (Barcelona), Sergio Ramos (Real Madrid)
Midfielders: Sergio Busquets (Barcelona), Ignacio Camacho (Malaga), Santi Cazorla (Arsenal), Cesc Fabregas (Chelsea), Raul Garcia (Atletico Madrid), Isco (Real Madrid), Koke (Atletico Madrid)
Forwards: Paco Alcacer (Valencia), Jose Callejon (Napoli), Alvaro Morata (Juventus), Nolito (Celta Vigo), Pedro (Barcelona)
