In order to establish an accurate and rapid method for identifying and detecting Vibrio parahaemolyticus（VP）and Aeromonas hydrophila（AH），based on the conserved region sequences of VP Tlh and AH aerA genes published by GenBank，two pairs of specific primers were designed and synthesized，followed by optimization of the reaction system and procedure，a dual PCR assay was established for simultaneously detecting VP and AH，and tested for its specificity and sensitivity. The results showed that the amplified bands of VP Tlh and AH aerA genes were 500 and 760 bp，respectively；the optimal concentrations of the primers were 7.5 and 10.0 nmol/L，respectively in the reaction system，and the optimal annealing temperature was 56.8 ℃ in the reaction procedure；the minimum detectable bacterial concentrations for VP and AH by this method were both 1.2×103 CFU/mL；it failed to crossly react with Bacillus licheniformis，Microbacillus，Bacillus megaterium，Bacillus cereus，Proteus vulgaris，Aeromonas viridis，Citrobacter brucei，Aeromonas guinea-pig，Vibrio cholerae，Vibrio mimicus，Aeromonas jeanensis，but with a specific positive amplification for VP and AH. In conclusion，a dual PCR assay with strong specificity and high sensitivity was established for rapid differential diagnosis of VP and AH，which could technically support rapid diagnosis，monitoring and effective prevention and control of aquatic animal diseases caused by bacterial pathogens such as VP and AH.