The 36-year-old is the most capped player for La Roja, having featured for the national side on 180 occasions, and his form for Paris Saint-Germain this season was expected to earn him a spot in the selection.
However, Spain boss Luis Enrique has left the defender out of the squad, with Eric Garcia, Hugo Guillamon, Pau Torres and Aymeric Laporte chosen ahead of him for the competition in Qatar.
There is also no space for Liverpool midfielder Thiago Alcantara, but there are five Premier League players in the squad - Laporte, Cesar Azpilicueta, Rodri, David Raya and Robert Sanchez.
David de Gea is also not included, but the goalkeeper's absence is not a surprise despite his strong form for Manchester United during the 2022-23 campaign.
There had been question marks raised over Ansu Fati's involvement, with the 20-year-old left out of Spain's most recent squad due to his struggles for starts in the early stages of the season.
However, the Barcelona forward, who has netted once in four appearances for La Roja, has been named in the selection.
In midfield, Sergio Busquets is included, while Koke, Rodri, Marcos Llorente, Pedri, Gavi and Carlos Soler are also options for Enrique in that area of the field.
Somewhat surprisingly, the Spain head coach has decided to name eight forwards in the squad, including youngsters Yeremi Pino and Nico Williams.
Alvaro Morata, Ferran Torres, Marco Asensio, Dani Olmo and Pablo Sarabia will also all be attacking options for La Roja during the tournament in Qatar.
Spain famously won the World Cup in 2010, but they have found it difficult to impress in their last two tournaments, being eliminated in the group stage in 2014 and only reaching the round of 16 four years later.
La Roja will compete in Group E this summer alongside Costa Rica, Germany and Japan.
Spain's tournament opener is against Costa Rica on November 23, before facing Germany four days later, ahead of their final game in the section against Japan on December 1.
Spain squad for World Cup:
Unai Simon (Athletic Club), Robert Sanchez (Brighton & Hove Albion), David Raya (Brentford); Dani Carvajal (Real Madrid), Cesar Azpilicueta (Chelsea), Eric Garcia (Barcelona), Hugo Guillamon (Valencia), Pau Torres (Villarreal), Aymeric Laporte (Manchester City), Jordi Alba (Barcelona), Jose Gaya (Valencia); Sergio Busquets (Barcelona), Rodrigo (Manchester City), Gavi (Barcelona), Carlos Soler (Paris Saint-Germain), Marcos Llorente (Atletico Madrid), Pedri (Barcelona), Koke (Atletico Madrid); Ferran Torres (Barcelona), Nico Williams (Athletic Bilbao), Yeremi Pino (Villarreal), Alvaro Morata (Atletico Madrid), Marco Asensio (Real Madrid), Pablo Sarabia (Paris Saint-Germain), Dani Olmo (RB Leipzig), Ansu Fati (Barcelona)