Abstract: The objective of this study is to develop a multiplex PCR assay that can simultaneously detect Salmonella spp.,Staphylococcus aureus,Listeria monocytogenes and Cronobacter spp.which with high detection rate in dairy products.Species-specific PCR primers were designed based on ompC gene for Salmonella spp.,hly gene for Listeria monocytogenes,nuc gene for Staphylococcus aureus and gyrB gene for Cronobacter spp..Developing and optimizing the multiplex PCR reaction system.The system were applied in the actual sample analysis after evaluating the specificity and sensitivity.The detection limit of Salmonella spp.template was 10^-6 ng/μL Listeria monocytogenes and Cronobacter spp.template were 10^-3ng/μL and Staphylococcus aureus template was 10^-2ng/μL.1 CFU/mL bacteria could be identified after pre-enrichment step.The multiple PCR method is specific,efficient and reliable for the detection of Salmonella spp.,Staphylococcus aureus,Listeria monocytogenes and Cronobacter spp.,and have good application prospects.