The aim of the study was explore a set of new detective targets in pathogenic Aeromonas veronii which was more specific than others at present, to develop a new duplex PCR method which could effectively detection the pathogenic A. veronii. Comparison analysis of the 16S rRNA gene sequences of Aeromonas spp. was used to explore A. veronii specific targets, which were then evaluated and used to design specific primers, and select the conservative primers of aerolysin genes in Aeromonas spp. to explore pathogenic targets. PCR parameters were optimized, its reaction system was developed, and its sensitivity and specificity were checked. 12 Aeromonas stains and 10 non-Aeromonas stains were tested for pathogenic A. veronii by duplex PCR method. The duplex PCR can clearly identify A. veronii from Aeromonas species, and can identify aerolysin-producing stains of A. veronii. The detection limit of genomic DNA by the duplex PCR was 1.35×10?3 mg/L. A novel duplex PCR method was successfully developed in this study, which could effectively detect pathogenic A. veronii with high accuracy and sensitivity.