Abstract: Cholesterol oxidase is a bacterial FADdependent flavoprotein that efficiently oxidates steroidal 3βhydroxy group to catalyze the first step in cholesterol metabolism. Cholesterol oxidase is an enzyme with great commercial value and has been used in biocatalysis for producing a variety of steroids, as an insecticidal protein against the cotton boll weevil larvae in agriculture and, in particular, as a diagnostic enzyme for determining serun levels of cholesterol. These important applications promote the researchers to isolate the cholesterol oxidase producers, purify the enzyme and reveal the relationship between structure and function. This review focused on the recent progress about the microbial species of producing cholesterol oxidase, the type and structure, the catalytic mechanism and the application of cholesterol oxidase. The key of future research was prospected.