Isco has made just five starts for Madrid in LaLiga this season, despite the club's domestic struggles, and his lack of playing time has seen him omitted from a much-changed selection.
His Madrid team-mates Sergio Reguilon and Nacho Fernandez have also been left out as have Atletico Madrid trio Diego Costa, Koke and Saul Niguez.
Uncapped quartet Sergio Canales, Sergi Gomez, Jaime Mata and Fabian Ruiz have all been included while there are returns for Jesus Navas and Iker Muniain.
Experienced Sevilla winger Navas has not played for his country in a competitive fixture since 2013 while Athletic Bilbao midfielder Muniain won his only cap a year earlier.
Defender Gomez is rewarded for a strong season with Sevilla, as is Canales with Real Betis while Ruiz has impressed in Serie A with Napoli.
Mata, 30, has scored 13 league goals in Getafe's bid for Champions League qualification, the club sitting fourth in the table heading into Matchday 28.
Spain's European Championship qualifying programme begins on March 23 when they host Norway in Valencia before travelling to Malta three days later.
Spain squad in full: David de Gea (Manchester United), Kepa Arrizabalaga (Chelsea), Pau Lopez (Real Betis); Sergi Roberto (Barcelona), Sergi Gomez (Sevilla), Sergio Ramos (Real Madrid), Mario Hermoso (Espanyol), Inigo Martinez (Athletic Bilbao), Jose Gaya (Valencia), Jordi Alba (Barcelona), Juan Bernat (Paris Saint-Germain); Jesus Navas (Sevilla), Sergio Busquets (Barcelona), Rodrigo (Atletico Madrid), Dani Ceballos (Real Madrid), Fabian Ruiz (Naples), Dani Parejo (Valencia), Sergio Canales (Betis); Marco Asensio (Real Madrid), Rodrigo (Valencia), Alvaro Morata (Atletico Madrid), Iker Muniain (Athletic Bilbao), Jaime Mata (Getafe).