A simple method of enzyme immobilization was investigated, which is useful for development of enzyme electrodes based on polyvinylferrocenium perchlorate coated Pt electrode surface. Enzymes were incorporated into the polymer matrix via ion exchange process by immersing polyvinylferrocenium perchlorate coated Pt electrode in enzyme solution for several times. Choline and acetylcholine enzyme electrodes were developed by co-immobilizing choline oxidase and acetylcholinesterase in polyvinylferrocenium perchlorate matrix coated on a Pt electrode surface. The amperometric responses of the enzyme electrodes were measured at +0.70 V versus SCE, which was due to the electrooxidation of enzymatically produced H2O2. The effects of the thickness of the polymeric film, pH, temperature, substrate and enzyme concentrations on the response of the enzyme electrode were investigated. The optimum pH was found to be pH 7.4 at 25degreesC. The steady-state current of these enzyme electrodes were reproducible within +/- 5.0% of the relative error. Response time was found to be 30-50 s and upper limit of the linear working portions was found to be 1.2 mM choline and acetylcholine concentrations in which produced detectable currents were 1.0 x 10(-6) M substrate concentrations. The apparent Michaelis-Menten constant and the activation energy of this immobilized enzyme system were found to be 1.74 mM acetylcholine and 14.92 kJ mol(-1), respectively. The effects of interferents and stability of the enzyme electrodes were also investigated. (C) 2003 Elsevier B.V. All rights reserved.