The cholesterol esterase and its oxidase were respectively immobilized onto sol-gel(tetraethyl orthosilicate)and then an enzymatic reaction column was prepared. Hydrolysis of cholesterol ester took place in the presence of cholesterol esterase to form cholesterol under catalysis of cholesterol oxidase, and Cholesterol was oxidized to produce hydrogen peroxide which reacted with luminal in the presence of simulated enzyme hemoglobin to result in the emission of light. Thereupon, a chemiluminescence system combined with flow injection technology by sol-gel immobilized enzyme was developed for the determination of cholesterol including free and total amount in human serum. The CL intensity was well linear with the concentration of cholesterol;liner range of total and free cholesterol was 1.01×10~(-6)～2.02×10~(-4) mol/L with a detection limit of 7.5×10~(-7) mol/L) and 5.0×10~(-8)～2.18×10~(-5) mol/L) with a detection limit of 5.0×10~(-9) mol/L respectively. Contrasted the method) with the biochemical analyzer (TBA-120FR), both the methods had no significant difference. This method has been successfully applied for the detection of cholesterol in clinic serum samples.