Abstract: On the basis of diagnosis kit for pathogenic Aeromonas, ECPase54 produced by Aeromonas hydrophila (Ah) was detected by DotELISA. On the other hand, the 16S rRNA and aerolysin of pathogenic Aeromonas hydrophila (PAh) were detected with PCR. Furthermore the skimmed milk plate tests were processed. 72 Aeromonas isolates were detected with the 4 different methods. The positive rate of 72 isolates was 90.3%（65/72）with DotELISA method , 75%（54/72）with skimmed milk plate method, 94.4%（68/72）with aer gene PCR method, 81.9%（59/72）with 16S rRNA PCR method. Compared DotELISA and the other three methods(skimmed milk plate, aer gene PCR and 16S rRNA PCR), the coincidence rate was 79.2%（57/72）, 91.6%（66/72）, 81.9%（59/72），respectively. The 72 isolates were repeatedly detected with DotELISA in 2, 4, 6, 12 and 18 months after preparing of the antiECPase54 rabbit serum, and the similarly results were got. In conclusion, this DotELISA method is sensitive, specific and practical. It can be used as a method for rapid diagnosis of PAh in clinic.