[Objective]Based on pirA and pirB gene probe,conducted an assay of fluorescence in situ hybridization (FISH),to detect the Vibrio parahaemolyticus which can cause acute hepatopancreatic necrosis disease (AHPND).[Methods]In silico analysis by BLAST and ProbeCheck showed that the designed probe targeting the pirA and pirB gene was suitable for specific identification of V.parahaemolyticus.The specificity and sensitivity of the probe pirA and pirB were verified by their differently reacting to 18 strains of 9 Vibrio species and 14 non-Vibrio strains of 14 representative species.The FISH assay can identify 20 V.parahaemolyticus positive samples from selectively enriched cultures of 81 samples selected from AHPND outbreak area.[Results]Combined with the results obtained by biochemical identification and PCR,we conclude that FISH can be used to detect AHPND pathogenic V.parahaemolyticus in shrimp and water samples.[Conclusion]This research will provide additional tools for a better understanding of the risk these environmental organisms pose to aquatic product.