The 37-year-old, Argentina's second most-capped player, informed his side Racing Club of his decision to hang up his boots on Friday.
Throughout his lengthy career, Ayala plied his trade at Napoli, AC Milan, Valencia and Real Zaragoza among other outfits.
On the international stage, he won 115 Argentina caps. Javier Zanetti is the country's most-capped footballer with 138 appearances.
Ayala featured at the 1998 and 2006 World Cups. He missed the 2002 competition after injuring himself during a warm-up against Nigeria.
He also played a major part in helping Valencia reach the Champions League final in 2001 and win two La Liga titles as well as one UEFA Cup.
Ayala, who was considered one of the best defenders on the globe, joined Argentine club Racing a year ago but couldn't secure a regular spot.
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