BACKGROUND: Hydrogen peroxide biosensors based on Poly(glycidyl methacrylate-co-3-thienylmethylmethacrylate)/ Polypyrrole [Poly(GMA-co-MTM)/PPy] composite film were reported. Poly(GMA-co-MTM) including various amounts of GMA and MTM monomers was synthesized via the radical polymerization. Enzyme horseradish peroxidase (HRP) was trapped in Poly(GMA-co-MTM)/PPy composites during the electropolymerization reaction between pyrrole and thiophene groups of MTM monomer, and chemically bonded via the epoxy groups of GMA. Analytical parameters of the fabricated electrodes were calculated and are discussed in terms of film electroactivity and mass transfer conditions of the composite films. RESULTS: The amount of electroactive HRP was found to be 1.25, 0.34 and 0.213 mu g for the working electrodes of Poly( GMA(30%)-co-MTM(70%))/PPy/HRP, Poly( GMA(85%)-co-MTM(15%))/PPy/HRP and Poly(GMA(90%)-co-MTM(10%))/PPy/HRP, respectively. Optimal response of the fabricated electrodes was obtained at pH 7 and an operational potential of -0.35 V. It was observed that effective enzyme immobilization and electroactivity of the composite films could be changed by changing the ratios of GMA and MTM fractions of Poly( GMA-co-MTM) based working electrodes. CONCLUSION: The amount of electroactive enzyme increases with increasing MTM content of the final copolymer. High operational stabilities of the biosensors can be attributed to the strong covalent enzyme linkage via the epoxy groups of GMA due to preventing enzyme deterioration and loss. A more convenient microenvironment for mass transfer was provided for the electrodes by higher GMA ratios. It is observed that mass transfer is dominated by the mechanism of electron transfer to obtain effective sensitivity values. This work contributes to discussions clarifying the problems regarding the design parameters of biosensors. (C) 2011 Society of Chemical Industry