Ramos dislocated a shoulder in Real's Champions League Group A match against Shakhtar Donetsk last month and although he played in Sunday's La Liga game at Atletico Madrid he is unable to feature for the European champions due to lingering pain, the Spanish football federation said on Tuesday.
Ramos is the latest to pull out following Villarreal midfielder Bruno Soriano, Real fullback Daniel Carvajal and Real Sociedad centre back Inigo Martinez, who all picked up injuries in La Liga action at the weekend.
They were replaced on Monday by Mario Gaspar of Villarreal and Athletic Bilbao pair Mikel San Jose and Xabier Etxeita.
Spain can make sure of their place at the tournament in France next year with a win against minnows Luxembourg, who are second-from-bottom in Group C, in Logrono on Friday.
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Vicente del Bosque's side then play their final qualifier against Ukraine in Kiev on Monday.
Spain have 21 points from eight matches, two ahead of Slovakia, who host Belarus on Friday. Ukraine are third on 16 points ahead of their match away to bottom side Macedonia.
(Reporting by Iain Rogers, editing by Pritha Sarkar)
