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.