A triple PCR method was established for the detection of Salmonella enteritidis, Staphylococcus aureus and Vibrio parahaemolyticus.The three strains were mixed cultured in SSV enrichment medium for 16 h, specific primers were designed for Salmonellaenteritidis invA gene, Staphylococcus aureus nuc gene and Vibrio parahaemolyticus collagenase gene, respectively, and the triple PCR system was established. By optimizing the primer concentration, annealing temperature and cycle times of the triple PCR system, thus optimal triple PCR system was established. The system was evaluated from three aspects: specificity, repeatability and sensitivity.The optimized result showed that, in the triple PCR system, the primers concentration of invA, nuc and collagenase were 10 nmol/L, 12.5 nmol/L and 12.5 nmol/L, respectively. The optimum annealing temperature was 55.6 ℃. The primers of the target bacteria had good specificity, and the detection sensitivity of the system was high. The sensitivity of the three strains in the triple PCR system was above 10~2 CFU/mL.The established triple PCR method has high specificity, high sensitivity and good repeatability, which provided a new technical support for the rapid detection of Salmonella enteritidis, Staphylococcus aureus and Vibrio parahaemolyticus.