Abstract: Objective:To establish a rapid multiplex polymerase chain reaction (PCR) method for the simultaneous detection of Salmonella spp., Shigella spp. and Enterohemorrhagic Escherichia coli O157∶H7. Methods:Three pairs of primers had been designed based on the invA gene in Salmonella spp., ipaH gene in Shigella spp.and uidA gene in Enterohemorrhagic Escherichia coli O157∶H7, single-factor experiment and L9 (34) orthogonal experiment were used to optimize multiplex PCR amplification system, and the sensitivity of multiplex PCR was also analyzed. Results:Fragments of 495, 620, 252 bp were obtained respectively. Under the optimized condition, the detection sensitivity of multiplex PCR for three pathogens was 104CFU/mL. This method had been applied to artificial experiment, the result was accurate and steady, which could be obtained within 5h.Conclusion:A simple, specific and sensitive multiplex PCR method had been established and it was valuable for rapid monitoring and diagnosis for Salmonella spp., Shigella spp. and Enterohemorrhagic Escherichia coli O157∶H7.