Immobilization of ascorbate oxidase (AO) in poly(3,4-ethylenedioxythiophene) (PEDOT)/multiwalled carbon nanotubes (MWCNTs) composite films was achieved by one-step electrochemical polymerization. The PEDOT/MWCNTs/AO modified electrode was fabricated by the entrapment of enzyme in conducting matrices during electrochemical polymerization. The PEDOT/MWCNTs modified electrodes were investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The experimental results showed that the composite films exhibited better mechanical integrity, electrochemical activity, higher electronic and ionic conductivity, and larger redox capacitance compared with pure PEDOT films, which would be beneficial to the fabrication of PEDOT/MWCNTs/AO electrochemical biosensors. The scanning electron microscopy studies revealed that MWCNTs served as backbone for 3,4-ethylenedioxythiophene (EDOT) electropolymerization. Furthermore, the resulting enzyme electrode could be used to determine L-ascorbic acid successfully, which demonstrated the good bioelectrochemical catalytic activity of the immobilized AO. The results indicated that the PEDOT/MWCNTs composite are a good candidate material for the immobilization of AO in the fabrication of enzyme-based biosensor. (C) 2011 Wiley Periodicals, Inc. J Appl Polym Sci 122: 1142-1151, 2011