Horse radish peroxidase, involved in enzyme-mediated luminol chemiluminescence, was immobilized on new polyamide membranes supplied in a preactivated form. In the presence of 1 × 10−4M luminol, H2O2can be detected between 1 × 10−8M and 1 × 10−4M by measuring the maximum intensity of light emitted. The use of soluble cholesterol oxidase (COD) as model enzyme, allowed a chemiluminescent determination of cholesterol. A compromise between the conditions for both enzymes activity expression, a sufficient light emission and a low background luminescence was accepted in the choice of the pH of assays fixed at 8. The chemiluminescent signal integrated during 150 sec was linear with cholesterol in the range 2.5 × 10−8M - 2.5 × 10−5M. With maximum light intensity measurements, a linear calibration graph was obtained from 1 × 10−8M to 2.5 × 10−4M but with a response-time depending on the measured concentration.