Background: Staphylococcus aureus is one of the main causes of hospital-acquired infections, which can cause skin and soft-tissue infections, systemic infections and toxemic syndromes. The development of multiple drug resistance to this organism is posing serious threat to inpatients,these bacteria also have plenty of virulence factors which contribute to infection. \nObjectives: To determine antimicrobial susceptibility pattern of S. aureus isolates from hospitalized patients and their virulence factors and biofilm formation at two Chinese University Affiliated Hospital.\nMethods: Swabs for culture of S. aureus were obtained from patients hospitalized in two hospitals in China from June 2013 to May 2014, and 135 isolates were collected. All the strains were analyzed with phenotypic assays in order to evaluate the virulence factors and the susceptibility against antibiotics. \nResults: In 135 strains of S. aureus,113 strains of β-lactamase were positive which probably accounted for the 100% resistance obtained for both Ampicillin and Penicillin. S. aureus isolates tested for antibiotic susceptibility showed high antibiotic resistance to Ampicillin, Penicillin, Amoxicillin, Nalidixic acid, and Ceftriaxone, mid resistance to Tobramycin, Neomycin, Erythromycin, Tetracycline, Gentamicine, Streptomycin, Kanamycin, Oxacillin and Co-trimoxazole, low resistance to Norfloxacin, Cephalexin, Ciprofloxacin, Rifampicin, Clindamycin and no resistance to Vancomycin. All S. aureus isolates were positive for proteases gelatinase and elastase production. The rate of positiveness for phospholipase, lipase and DNase tests were 72.6%, 75.6% and 91.1%. 32 out of 135 tested strains were beta-hemolytic (23.7%), 58 out of 135 tested strains were alpha-hemolytic (42.9%) and the other 45 strains can produce alpha- and beta-haemolysis (33.3%). 61.5% isolates were found to be positive for biofilm production by standard tube method, while 45.2% were found to be positive biofilm producers by congo red agar method.\nConclusions: On in vitro sensitivity testing, ampicillin, penicillin and amoxicillin were the least effective. Norfloxacin, cephalexin, ciprofloxacin, rifampicin, clindamycin and vancomycin were the most effective antibiotics. Clinical S. aureus isolates were producers of a variety of extracellular hydrolytic enzymes and can form biofilm, which might have an important role in the pathogenesis of infections and drug resistance.