The 25-year-old midfielder has been a regular starter for United since his switch from Stamford Bridge last month but he has failed to catch the eye of Spain boss Vicente del Bosque.
Del Bosque has also omitted Chelsea striker Fernando Torres from his 22-man squad for the high-profile friendly international against Italy in Madrid on Wednesday.
The duo now face a struggle to impress Del Bosque ahead of the World Cup this summer as this is his last game before he names his preliminary list of 30 players for Brazil in May, although the coach tweeted: "This is a list only for the match against Italy. It doesn't mean anything for Brazil 2014."
Juventus striker Fernando Llorente, who has been in fine form in Serie A, and Atletico Madrid's David Villa are other notable omissions, while Barcelona centre-back Gerard Pique is absent due to injury.
Atletico Madrid striker Diego Costa could make his debut for Spain at the Vicente Calderon - his home ground - after scoring 27 times in all competitions this season.
Costa completed the nationalisation process that allowed him to switch allegiances from Brazil to Spain in time for La Roja's friendlies against Equatorial Guinea and South Africa last November, but was forced to miss both games due to injury.
Bayern Munich midfielder Thiago Alcantara is rewarded for his impressive form for the Bundesliga champions with a call-up to the senior squad for the first time this season.
Chelsea full-back Cesar Azpilicueta is also included for the first time since the Confederations Cup ahead of Real Madrid's Alvaro Arbeloa.
Del Bosque said: "We think Azpilicueta is playing well at Chelsea and we will take a closer look at him.
"The same with Thiago. He is playing for Bayern Munich, one of the best teams in Europe, and he is in the starting line-up despite the high degree of competition."
Manchester City duo David Silva and Alvara Negredo also feature in the squad.
Meanwhile, it has also been confirmed that Spain will play their final warm-up game before the World Cup against El Salvador in Washington, D.C. on June 7.
Goalkeepers: Iker Casillas (Real Madrid), Pepe Reina (Napoli), Victor Valdes (Barcelona).
Defenders: Juanfran (Atletico Madrid), Raul Albiol (Napoli), Sergio Ramos (Real Madrid), Javi Martinez (Bayern Munich), Jordi Alba (Barcelona), Cesar Azpilicueta (Chelsea).
Midfielders: Xavi (Barcelona), Sergio Busquets (Barcelona), Xabi Alonso (Real Madrid), Koke (Atletico Madrid), Andres Iniesta (Barcelona), Santi Cazorla (Arsenal), Cesc Fabregas (Barcelona), Thiago Alcantara (Bayern Munich), David Silva (Manchester City).
Forwards: Alvaro Negredo (Manchester City), Jesus Navas (Manchester City), Pedro (Barcelona), Diego Costa (Atletico Madrid).