The 20-year-old, who made his national team debut last September, was overlooked by coach Javier Aguirre despite playing the full 90 minutes of Mexico's 5-1 friendly win over Gambia yesterday.
Defensive duo Juan Carlos Valenzuela (America) and Adrian Aldrete (Morelia) were the other two players to miss out.
"The Mexican Football Federation is grateful to Jonathan Dos Santos Ramirez for their dedication, discipline, professionalism and commitment shown during the preparations with the national team," read a statement on the governing body's official website.
While Dos Santos missed out there was better news for his brother, Giovani, who was included after spending last season on loan at Galatasaray under his former Barça boss Frank Rijkaard.
Manchester United-bound striker Javier Hernandez was included following his double in the friendly against Gambia in Germany. That match was Mexico's last before the FIFA World Cup finals where they will play hosts South Africa, France and Uruguay in Group A.
Mexico's 23-man FIFA World Cup squad: Oscar Perez (Chiapas), Guillermo Ochoa (America), Luis Ernesto Michel (Guadalajara); Rafael Marquez (Barcelona), Ricardo Osorio (Stuttgart), Francisco Rodriguez, Carlos Salcido (both PSV Eindhoven), Hector Moreno (AZ Alkmaar), Paul Aguilar (Pachuca), Efrain Juarez (Pumas UNAM), Jonny Magallon (Guadalajara), Jorge Torres Nilo (Atlas); Andres Guardado (Deportivo La Coruna), Gerardo Torrado (Cruz Azul), Israel Castro (Pumas UNAM), Giovani dos Santos (Galatasaray, on loan from Tottenham); Pablo Barrera (Pumas UNAM), Adolfo Bautista, Alberto Medina (both Guadalajara), Cuauhtemoc Blanco (Veracruz), Carlos Vela (Arsenal), Javier Hernandez (Manchester United), Guillermo Franco (West Ham).
*Please note that squad lists only become official after they have been submitted to and announced by FIFA.