Objective To develop Real-Time fluorescence PCR assay for detecting Salmonella, Shigella, Listeria monocytogenes, EHEC O157∶H7, Staphylococcus Aureus, and apply this assay in food borne pathogens monitoring. Methods DNA of the strains and sample were extracted on boiling lyses method after cultivation over night. The specificity of the Real-time PCR assay was tested by using the PCR kit to amplified previous DNA. And the detection rate of the Real-Time PCR assay and the culture method were obtain by comparison with the two different method in testing 890 food samples during 2006-2007. Results Coincidence rate of the detection of 19 standards staid on Real-Time PCR assay was 100%. Testing the isolates from food monitoring early on Real-Time PCR assay showed the coincidence rate of 96.61% in Salmonella, 92.30% in Listeria monocytogenes, and 100% in the pathogens of Shigella, EHEC O157∶H7 , Staphylococcus Aureus. Comparison of the Real-Time PCR assay with the traditional detection method in testing 890 food samples showed Real-Time PCR assay in detection rate were slightly higher than traditional methods. Though the difference is insignificant（P＞0.05）, Real-Time PCR assay can get the result in 3-36 hour. Conclusion We consider that this real-time PCR assay is rapid, sensitive and specific. It can be provide new feasible technology for the rapid diagnosis of pathogens in food poisoning surveillance and safeguard food safety during major assembly.