Abstract: A Fiber optic biosensor responding to both of cholesterol esters and free cholesterol is prepared by covalently coupling cholesterol esterase, cholesterol oxidase and horseradish peroxi-dase to bovine serum albumin via glutaraldehyde and making a membrane to end of fiber-optic handle.This sensor is based on cholesterol esterase and catalyzes the hydrolysis of cholesterol esters to free cholesterol, then the cholesterol oxidase in the prescence of oxygen oxidizes the free cholesterol to 4-cholestene-3-one and hydrogen peroxide, which reacts with luminol under the catalysis of horseradish peroxidase to produce chemiluminescence.The linear range for cholesterol is 0.5-20 μg/mL.The detection limit is 0.1 μg/mL.The response time is 2 min.The life time of membrane is longer than 2 months.This biosensor has been applied to determining the total cholesterol and free cholesterol in serum with specificity.

Key words: Fiber optic biosensor, Enzyme optrode, Chemiluminescence, Cholesterol