An amperometric vitamin C biosensor was facilely fabricated by the immobilization of ascorbate oxidase (AO) on polypyrrole (PPy)multiwalled carbon nanotubes (MWCNTs) composites with a one-step electrodeposition technique in a 0.05M phosphate buffer solution (pH 6.5). The cyclic voltammetry, IR spectral analysis, electrochemical impedance spectroscopy, and scanning electron microscopy measurements indicated that AO was successfully immobilized on the PPyMWCNT composites. The optimization of the biosensor parameters, including the working potential, pH, and temperature, was investigated in detail. The proposed biosensor showed a linear range of 5 x 10-5 to 2 x 10-2 M with a detection limit of 0.3 mu M, a sensitivity of 25.9 mA mM-1 cm-2, and a current response time less than 20 s under the optimized conditions. The apparent MichaelisMenten constant together with the apparent activation energy indicated that the proposed biosensor exhibited a high bioaffinity and a good enzyme activity. In addition, the biosensor also showed good operational and storage stabilities. (c) 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012