An amperometric choline biosensor was constructed by immobilizing choline oxidase (ChO) on poly(2-hydroxyethyl methacrylate) (PHEMA)-grafted Teflon (polytetrafluoroethylene, PTFE) film. Grafting was achieved by gamma irradiation. PHEMA-grafted Teflon films were activated with epichlorohydrin or glutaraldehyde to achieve covalent immobilization of enzyme onto the film. To decrease the diffusional barrier caused by the enzyme-immobilized film, the film was stretched directly on the electrode. The PHEMA-grafted Teflon film, therefore, had to have appropriate mechanical properties. Glucose oxidase (GOD) was used in the determination of optimum immobilization conditions, then these were applied to ChO. With GOD, the effect of activation type and film position in electrode on enzyme activity was studied and the highest catalytic activity was obtained when the enzyme was immobilized using glutaraldehyde and the film was stretched over the electrode surface. Further studies revealed that the films activated with glutaraldehyde, immobilized in 2 mg/mL ChO concentration, and stretched directly on the electrode were suitable (specific activity, 0.427 +/-0.068 U mg(-1)) for use in the choline biosensor. The linear working range of this biosensor was found to be 52-348 mu M, with a 40 +/- 5 mu M minimum detection limit. The response of the sensor, however, decreased linearly upon repeated use. (c) 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 3469-3477, 2007.