Cholesterol oxidase is a profound enzyme that recently gained much attention, especially for its numerous therapeutic and biotechnological applications, as a rapid and sensitive assay for cholesterol estimation in various clinical samples, by catalyzing the conversation of cholesterol into cholest-4-en-3-one and reducing oxygen to hydrogen peroxide. The immobilized enzyme has been commonly used as an electrode in the colorimetric biosensors of cholesterol estimation, with a high sensitivity and selectivity for arteriosclerosis diagnosis, preparation of the ketosteroids from the corresponding ones, and steroids analysis in food samples. This enzyme was partially purified and characterized from various microbial sources. Nevertheless, the availability, structural stability, and catalytic efficiency of the completely purified cholesterol oxidase remain the major challenges. So, the objective of this review was to give an insight into the different sources, biochemical and catalytic properties of cholesterol oxidase from the literature study, in addition to the possible approaches to increase its reusability and operative stability via different immobilization techniques.