Abstract: In this study to isolate cholesterol oxidase (Cho) producing microorganism, a bacterium with high ability to produce Cho was found. It was identified with morphological, biochemical and molecular methods as a new species of Rhodococcus. Rhodococcus sp. PTCC 1633 has both extra-cellular and intracellular Cho. It was cultured in optimized condition and extra-cellular Cho concentrated by Three Phase Partitioning (TPP) with a recovery of 85%. The concentrated enzyme was purified by one step ion exchange chromatography. On SDS-PAGE the purified enzyme showed a molecular weight of about 55 kDa. The enzyme was active in a wide rang of pH and temperature with an optimum pH and temperature of 7.0-7.5 and 40°C, respectively, for activity. The Km value for this enzyme was 15 μM. A thermal stability experiment showed high stability at 40°C in 24 h.