Ninety one samples were collected from different clinical and environmental sources to isolate cholesterol oxidase producing Pseudomonas sp. , from these samples, 110 isolate were obtained, sixty of them were belong to Pseudomonas sp. Since they have the ability to grow on citrimide agar medium. Results of screening of these isolates for cholesterol oxidase production showed that Pseudomonas sp. H48 was the most efficient among the tested isolates in enzyme production since the activity of crude enzyme in culture filtrate was 1.71 U/ml. Identification of this isolate using Api 20 E system indicated that it is Pseudomonas aeruginosa. Optimum conditions for cholesterol oxidase production by P. aeruginosa H48 were studied using Lauria _ bretani broth as a production medium. Results showed that the optimum conditions for enzyme production includes the use of glucose as a sole source for carbon and energy in a concentration of 2.5 meat extract as a nitrogen source in a concentration of 0.5%, 0.018% of K2HPO4 and 0.006% of KH2PO4 as a phosphate source, while the optimum pH for production medium was 7.0, optimal conditions also includes the incubation at 32°C for 48hr. in a shaker incubator at 150 rpm. Under these conditions cholesterol oxidase specific activity was 3.84 U/mg. Cholesterol oxidase was partially purified, first by precipitation with ammonium sulphate (70% saturation), followed by dialysis and purification by ion exchange chromatography using CM-cellulose column. Activity of cholesterol oxidase was appeared in the second peak of elution hence the enzyme activity was reaches to 4.4 U/ml. Optimum pH and temperature for enzyme activity was studied. Results showed that the optimum pH and temperature for enzyme activity was pH 7.0 and 35°C respectively, while the optimum pH and temperature for enzyme stability was pH 6.5 and 40°C respectively.