Abstract: Objective: In order to establish a effective method to detect Vibrio cholera,Vibrio Parahaemolyticus and Listeria monocytogenes by triple real- time PCR.Methods: Specific primers and probes were designed,which based on the zot toxin gene of CTX in Vibrio cholera,tl gene of TDH in Vibrio Parahaemolyticus and hly gene of Listeria monocytogenes. What is more,the performance of the test kit was established and evaluated. Results: The correlation coefficients of standard curve linears for Vibrio cholera,Vibrio Parahaemolyticus and Listeria monocytogenes were 0.9998,0.999 and 0.9963. The intra- assay coefficient variables of the three pathpgens at 10~5 cfu / m L and 103 cfu / m L concentration logarithm were 0.40%,0.83%,0.49% and0.75%,0.81%,0.64%; 0.76%,1.03%,0.62% and 1.06%,0.72%,1.26%; 0.62%,0.51%,0.81% and 1.38%,0.76%,2.34%; inter- assay coefficient variables were 0.58% and 0.76%,0.83% and 1.01%,0.66% and 1.58%. The sensitivity indicated that the lowest accurate quantitative concentrations of Vibrio cholerae,Vibrio parahaemolyticus,Listeria monocytogenes were 23,16,35 cfu / m L. Conclusion: A rapid detection method for testing the three pathogens of aquatic products was established.