[Objective] The mastitis of dairy cows in a large-scale pasture in Xinjiang was more serious, and it had not been cured for a long time. Explore the cause of the disease and find a reasonable response. [Method] In this study, typical dairy cow mastitis materials were collected, and they were separated, identified and tested for drug susceptibility. The results of isolation and purification of pathogenic bacteria showed that the pathogenic bacteria of mastitis in dairy cows were mainly caused by the mixed infection of Escherichia coli, Staphylococcus aureus, Clostridium and other bacteria. The results of antimicrobial susceptibility test showed that Escherichia coli was highly sensitive to cotrimoxazole, kanamycin, enrofloxacin and ciprofloxacin, among which ciprofloxacin was extremely sensitive. Staphylococcus aureus was extremely susceptible to cotrimoxazole, kanamycin, enrofloxacin, and chloramphenicol.Clostridium was highly susceptible to chloramphenicol, ceftriaxone, ampicillin. [Conclusion] In this study, Escherichia coli, Staphylococcus aureus and Clostridium were isolated from milk samples from mastitis, and it was recommended that the farm should take isolation measures for the diseased cows and use tilmicosin, ceftiofur sodium, and clostridial vaccine for combined treatment.