Abstract: A novel cholesterol oxidase is disclosed. The enzyme according to the present invention has the following properties: acts on cholesterol to convert it to cholest-5-en-3-one; acts 3&bgr;-sterols but not on 3&agr;-hydroxysteroids; optimum pH: 5.0-8.5; and stable pH: 4 to 11. This enzyme carries out the oxidation reaction at high velocity at low substrate concentrations, reacts in a broad pH range, is heat resistant, and reacts at high reaction velocity even in the presence of an organic solvent. The enzyme according to the present invention is useful as a reagent for measuring cholesterol concentration, as a composition for the extermination of harmful insects, and as a bleaching agent.