【Objective】The present study was conducted to investigate the isolation and purification method of extracellular cholesterol oxidase （COD） from Paenibacillus to provide technical support for Paenibacillus’ production and application. 【Method】The fermentation of Paenibacillus was treated through circumrotate evaporation evaporation， sucrose diallysis， DEAE－cellulose ion exchange column  and Sephadex－G75 gel filtration column chromatography to purify target protein and determining its fundamental enzymological characteristics. 【Result】After Paenibacillus  fermentation broth was processed by DEAE－cellulose ion exchange column and Sephadex－G75 gel filtration column chromatography， the purified enzyme specific activity reached 6.83 U/mg， the recycling rate reached 35.6% and purification multiple was 13.4 times. The purified enzyme demonstrated to be a single band on SDS-PAGE， and its molecular weight was about 58 kDa. Applied to cholesterol in different concentrations， Km of the purified COD was 3.3×10-5 mol/L using Lineweaver Burk mapping. The result of thin layer chromatography indicated that the product was cholesteric-4-olefin-3-ketone and the purified enzyme was COD. 【Conclusion】Electrophoretical purity extracellular COD from Paenibacillus was obtained through isolation and purification， and it is worthy of more application and studies for its positive affinity to cholesterol.