This book constitutes the refereed proceedings of the 31st International Symposium on String Processing and Information Retrieval, SPIRE 2024, held in Puerto Vallarta, Mexico, during September 23–25, 2024. The 22 full papers and 4 short papers presented in this volume were carefully reviewed and selected from 41 submissions. The papers reflect the continuation of the long and well-established tradition of encouraging high-quality research at the broad nexus of string processing, information retrieval, and computational biology.