In this study, a novel amperometric glucose biosensor was developed by immobilizing glucose oxidase (GOX) by cross-linking via glutaraldehyde on electrochemically polymerized polypyrrole-poly (vinyl sulphonate) (PPy-PVS) films on the surface of a platinum (Pt) electrode. Electropolymerization of pyrrole and poly (vinyl sulphonate) on the Pt surface was carried out with an electrochemical cell containing pyrrole and poly (vinyl sulphonate) by cyclic voltammetry between -1.0 and + 2.0 V (vs.Ag/AgCl) at a scan rate of 50 mV/s upon the Pt electrode. The amperometric determination was based on the electrochemical detection of H2O2 generated in enzymatic reaction of glucose. 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 optimum parameters were found to be 7.5 and 65 degrees C, respectively. The effect of working potential was investigated and optimum potential was determined to be 0.4 V. The operational stability of the enzyme electrode was also studied. The response of the PPy/PVS-GOX glucose biosensor exhibited good reproducibility with a relative standard deviation (RSD) of 2.48%. The glucose biosensor retained 63% of initial activity after 93 days when stored in 0.1 M phosphate buff er solution of pH 7.5 at 4 degrees C. With the low operating potential, the biosensor demonstrated little interference from the possible interferants.