The invention provides a composition used for detection of methicillin-resistant and / or tetracycline-resistant staphylococcus aureus. The composition used for detection of the methicillin-resistant and / or tetracycline-resistant staphylococcus aureus comprises a primer pair shown in SEQ ID No.1 / 2 and at least one of primer pairs shown in SEQ ID NO.3 / 4 and SEQ ID NO.5 / 6; meanwhile, an mPCR-DHPLC / electrophoresis detection kit and method are further constructed on the basis, and pathogenic staphylococcus aureus, MRSA and tetracycline-resistant staphylococcus aureus drug resistant strains in food are detected; and bacterial strain identification is carried out on staphylococcus aureus by utilizing the composition and the method which are disclosed by the invention, and MRSA and a tetracycline-resistant gene can be detected at the same time, so that staphylococcus aureus, MRSA and erythromycin-resistant staphylococcus aureus in a sample can be identified at the same time during one-step detection. The composition used for detection of methicillin-resistant and / or tetracycline-resistant staphylococcus aureus has the advantages of high speed and high throughput; and meanwhile, detection time is short, operation is simple, labour and cost can be greatly saved, and the rapid detection requirement can be met.