One hundred samples were collected from different body sites and lesions (UTI, wounds, and ear swabs….etc.) of in and out patients from both sexes who attended Al-Kadimyia teaching Hospital in Baghdad during the period from November-2010 until March-2011 for the isolation and identification of Staphylococcus aureus. Bacterial samples were identified by subjecting them to the standard laboratory procedures and the results showed that forty isolates out of the total of 100 were identified as Staphylococcus aureus.Semi quantitative screening on blood agar (containing 5% human blood) revealed that all isolates were hemolysin producer but in different efficiencies. Depending on the semi-quantitative screening and hemolytic assays isolate SW-14 of Staphylococcus aureus was the higher hemolysin producing isolate so it was selected as a candidate for the isolation, purification and characterization of hemolysin.Determination of the optimal conditions for hemolysin production including the optimum pH and temperature were performed, the results demonstrated that the best hemolysin production was in the pH near neutrality (pH 7-7.5) and in temperature of 35-40oC.The hemolysin was extracted as a crud enzyme from the liquid culture media (brain heart infusion broth (BHIB)) by cooling centrifugation at 7000 rpm at 4oC for 15 minutes, and after the extraction hemolysin was purified by many steps including: precipitation by ammonium sulphate with 50-75% saturation percentage, dialysis, ionic exchange chromatography by using DEAE-Cellulose, and gel filtration by using Sephadex G-100. The results declared that hemolysin was purified 135 fold with a yield of 1.16%.Furthermore, the results of purified enzyme characterization indicated that the molecular weight of hemolysin determined by gel filtration chromatography on Sephadex G-100 column was about 35000 daltons, while the optimum pH for enzyme stability was 7 and the optimum temperature for enzyme stability was between 25-35oC.