The club says Puyol, who had just been cleared to play before the Copa del Rey final earlier this week, will be unavailable after his troublesome right knee acted up again during training.
Barcelona was already without defenders Gerard Pique and Jordi Alba, striker Neymar and goalkeeper Victor Valdes.
Barcelona defender Marc Bartra has been included on its squad after being considered doubtful.
Barcelona needs to win to remain within four points of league leader Atletico Madrid.
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