To develop a multiplex PCR assay to detect Enterohemorrhagic Escherichia coli (EHEC), Vibrio parahaemolyticus (VP), Salmonella spp. and Listeria monocytogenes(LM) in innocuous poulthy and seafood simultaneously, four pairs of primers were designed from hemolysis gene (hlyAB) of EHEC, toxR gene of VP, invA gene of Salmonella spp. and iap gene of LM. They were detected by conventional PCR and multiplex PCR assay. DNA sequencing was used to examine PCR products. This multiplex PCR method was a highly specific, rapid and sensitive method which could simultaneously detect EHEC, VP, Salmonella spp. and LM. The detection level was 10~(3)cfu/mL.