We describe the effect of preparation conditions during electropolymerization such as enzyme concentration in the deposition solution and film thickness on the analytical performance of the resulting polypyrrole / glucose oxidase (PPy/GOD) film. The responses of the resulting PPy/GOD biosensor to glucose and ascorbate were characterized and optimized. It was found that the enzyme concentration and the film thickness have the equivalent effects on the response of the resulting biosensor and that several optimal sets of the two parameters exist. The investigations regarding the response characteristic and the suppression of interference clearly indicate that the performance of biosensor could be significantly improved by making the film thick at low enzyme concentration.