Cholesterol oxidase is a key enzyme during cholesterol metabolism. It could be potential used to detect the cholesterol level in serum in clinical trials. The cholesterol oxidase from Rhodococcus ruber (ChOG) was transformed into BL21(DE3) and Rosetta(DE3) for protein expression, and was induced at different conditions. The results showed that ChOG was overexpressed at 16℃ and 0.1 mmol/L IPTG. The activity reached 8.033 U/mg after purification with Ni-NTA superflow. The product cholest-5-en-3-one was characterized by TLC and HPLC, respectively. The structural analysis showed that Glu406, together with Arg408 and Glu261 played an important role during the dehydrogenation of cholesterol C3-OH, proton transfer, and isomerization.