Abstract: Staphylococcus aureus is one of main pathogens that causes the mastitis of dairy cow. Because the mastitis of dairy cow had higher incidence and it was difficult to be cured, so it brought great economic loss for the milk industry and had greater threatens for the food safety. Taking the isolated strains of mastitis-derived S. aureus as the research objects, their morphology on Congo red medium was observed. The drug sensitivity of 5 isolated strains of mastitis-derived S. aureus to 19 kinds of antibiotics was analyzed by using diffusion method. The results showed that 5 strains of S. aureus all had the ability of forming the biofilm. They were totally tolerant to common antibiotics of penicillin, streptomycin, ampicillin and achromycin, and they were moderately sensitive to vancomycin, ciprofloxacin, norfloxacin, amikacin and other antibiotics. The isolated strains were highly sensitive to 3 kinds of antibiotics （enrofloxacin, cefoxitin and cefotaxime）. The research results indicated that mastitis-derived S. aureus had stronger drug tolerance, and the drug tolerance might be related with the forming of its biofilm.