In this study, a novel amperometric glucose biosensor with immobilization of glucose oxidase on electrochemically polymerized polyaniline-polyvinylsulphonate (Pani-Pvs) films has been accomplished via the entrapment technique. Electropolymerization of aniline on the Pt surface of the Pt electrode was carried out at constant potential (0.75 V, vs. Ag/AgCl) using an electrochemical cell containing aniline and polyvinylsulphonate. Firstly, the optimum working conditions for preparing polyaniline-polyvinylsulfonate films were investigated. Determination of glucose was carried out by the oxidation of enzymatically produced H2O2 at 0.4 V vs. Ag/AgCl. The effects of pH and temperature were investigated and the optimum pH value was found to be 7.5. The storage stability and operational stability of the enzyme electrode were also studied. The results show that 75% of the response current was retained after 16 activity assays. The prepared glucose biosensor retained 80.6% of initial activity after 40 days when stored in 0.1 M phosphate buffer solution at 4 degrees C.