Aeromonas is one of the major pathogens which frequently cause the bacterial diseases in freshwater fish terrapin and crustaceans. Aeromonas hydrophila caused economic losses widely. In order to detect Aeromonas spp. and Aeromonas hydrophila,two pairs of specific primers were designed according to the glycerophospholipid-cholesterol acyltransferase (GCAT) gene of Aeromonas spp. and the 16S rRNA gene of Aeromonas hydrophila. So a duplex PCR assay was developed to amplify the 16S rRNA gene and the GCAT gene. The reaction conditions of the duplex PCR were optimized and PCR products were sequenced. Meanwhile,the specificity of duplex PCR was studied. Five Aeromonas hydrophila strains,seven Aeromonas strains and the other five strains of non Aeromonas were tested by duplex PCR. The results showed that the GCAT gene and the 16S rRNA gene could be amplified from Aeromonas hydrophila strains,but only the GCAT gene from the other Aeromonas species and neither from the other five strains. It could be concluded that the duplex PCR was specific and could be used in quick diagnose Aeromonas hydrophila and Aeromonas spp.