Sixty milk samples were collected from dairy cows with mastitis in Shanghai to study the antimicrobial resistance of Staphylococcus aureus. Staphylococcus aureus isolation, identification and antimicrobial resistance were investigated. Eight Staphylococcus aureus isolates (8/60, 13.33%) were obtained from the collected samples. The antimicrobial resistance test revealed that 100.00% of the isolates were resistant to penicillin G and ampicillin as well as cefalotin, cefmetazole and cefuroxime sodium, 50.00% were resistant to sulfamethoxazole, 37.50% were resistant to cefaclor, ceftizoxime, lomefloxacin, norfloxacin, tetracycline, gentamicin, erythromycin, clindamycin and chloramphenicol, 25.00% were resistant to ciprofloxacin and ofloxacin, and 12.50% were resistant to cefotaxime, ceftazidime and enoxacin. Four Staphylococcus aureus isolates had multi-resistance abilities.