In this paper, a new amperometric biosensor based upon a conducting polymer with anionic dopant-modified electrode was developed for use in the determination of glucose. Glucose oxidase enzyme was immobilized in polypyrrole-poly(sodium-4-styrenesulphonate) film using the entrapment method. Amperometric determination is based on the electrochemical detection of H2O2 generated in the enzymatic reaction of glucose oxidase. Glucose detection was carried out according to the oxidation of hydrogen peroxide at 0.4 V resulting from the enzymatic reaction on the biosensor surface. Some parameters that affect amperometric response current were investigated in terms of temperature, pH, and substrate concentration. The stability and reproducibility of the biosensor were analyzed. The interference effects were examined using amperometric response of the biosensor. The glucose detection ability of the biosensor was tested in a biological fluid (in blood). The film thickness, morphology, and water contact angle measurements of the polypyrrole-poly(sodium-4-styrenesulphonate) film were characterized by profilometer, atomic force microscope, and water contact angle device, respectively. For the biosensor in this study, the linear range 1 x 10(-8)-1 x 10(-3) M, optimum temperature 25 degrees C, optimum pH 8, K-m value 2.97 mM and V-max value 0.097 mu A was found. The results of the study show that the film can provide suitable biological application and electrochemical microenvironment for immobilization of the enzymes, making this material a perfect candidate for the production of extremely susceptible and selective glucose biosensors.