Objective To establish a multiplex PCR method to detect penicillinase gene, mecA gene and 16S rDNA simultaneously, and investigate the distribution of penicillinase gene, a kind of choloylglycine hydrolase, and mecA gene in foodborne Staphylococcus aureus strains. Methods The multiplex PCR method was established to detect penicillinase gene, mecA gene and 16S rDNA. Results 165 strains were detected penicillinase gene with overall positive rate 96.5%, while 9 strains were detected both mecA gene and penicillinase gene with overall positive rate 5.3%. Conclusion The multiplex PCR method which was rapid, convenient and precise, could be used for high throughput screening of penicillinase gene, mecA gene and 16S rDNA. Most strains of foodborne Staphylococcus aureus carry penicillinase gene, and a few of them are methicillin resistant.