Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae are important pathogens that pose a risk to human health. In this study, a PMA-mPCR assay was developed for detecting viable vibrios in aquatic foods. A total of 206 bacterial strains were used to evaluate the specificity. The results showed that this assay exhibited 100% specificity. The limit of detection (LOD) for the viable cells with or without PMA treatment was approximately 103 CFU/mL (1.05 × 103 CFU/mL with PMA, 1.5 × 103 CFU/mL without PMA) for V. parahaemolyticus, 103 CFU/mL (1.68 × 103 CFU/mL with PMA, 1.45 × 103 CFU/mL without PMA) for V. alginolyticus, and 103 CFU/mL (1.03 × 103 CFU/mL with PMA, 1.0 × 103 CFU/mL without PMA) for V. cholerae, reflecting that PMA treatment did not change the sensitivity. After 10 h of enrichment, the PMA-mPCR assay detected as low as 1.0 × 102 CFU/g viable V. parahaemolyticus, V. alginolyticus, and V. cholerae in spiked seafood. A total of 453 actual samples were used to assess the practical application of the assay, and the results indicated that target vibrios in seafood were accurately screened. This PMA-mPCR assay has potential as a promising tool for the rapid, reliable and simultaneous detection of viable vibrios in aquatic foods. © 2023 The Authors