A polymer-based amperometric enzyme electrode (Pt/PoPD/ChOx) was prepared for the amperometric detection of free cholesterol. Firstly, poly(o-phenylenediamine) (PoPD) polymer film was prepared in acetonitrile-water medium containing o-phenylenediamine (oPD) monomer and (+/-)-10-camphorsulfonic acid (HCSA) on Pt electrode by the use of cyclic voltammetry technique. Cholesterol oxidase (ChOx) enzyme was immobilized onto Pt/PoPD electrode surface. The determination of cholesterol was performed via monitoring of the oxidation current of enzymatically produced H2O2 at + 0.7 V vs. Ag/AgCl. Optimum buffer concentration, pH and working temperature were found as 0.05 mol L-1, 7.5 and 40 degrees C, respectively. The working range of enzyme electrode to cholesterol was 9.8 x 10(-3)-11 mu mol L-1 and response time 150 s. The effects of possible interferences present in serum samples on response of enzyme electrode were examined. The determination of total cholesterol in serum samples was performed by using proposed Pt/PoPD/ChOx enzyme electrode and results were in good agreement with those obtained by spectrophotometric method.