Abstract: To establish a multiple PCR assay for the effective and simultaneous detection of Cronobacter spp.,Salmonella spp. and Staphylococcus aureus in powdered infant formula(PIF). Three pairs of primers(OmpA,invAF1 and clfA)with high specificity were screened from nine previously reported pairs of primers. The specificity and sensitivity of the established multiple PCR assay was evaluated,and then the multiplex PCR assay was applied to artificially contaminated PIF for the detection of the three foodborne pathogens. The results indicated that the amplified fragment sizes of Cronobacter spp.,Salmonella spp. and Staphylococcus aureus were 469,638 and 796 bp,respectively which had highly specificity. The optimal annealing temperature of the multiplex PCR assay for was 55℃,the Mg²⁺ concentration of 2.00 mmol/L,and the concentration of primers of 400 nmol/L for each specific primer for the simultaneous detection of Cronobacter spp.,Salmonella spp. and Staphylococcus aureus in a single PCR tube. The specificity of the multiplex PCR assay was high,and the sensitivity of multiplex PCR assay was 10² CFU/mL for pure cultures under the optimized conditions. When the multiplex PCR assay was applied to artificially contaminated PIF,the detection limit of the multiplex PCR was 10³ CFU/g. The multiplex PCR assay was thus an accurate and specific method for the rapid detection of Cronobacter spp.,Salmonella spp. and Staphylococcus aureus in PIF.