An amperometric L-ascorbic acid (AA) biosensor fabricated by immobilizing ascorbate oxidase (AO) in poly(3,4-ethylenedioxythiophene) (PEDOT) and multi-walled carbon nanotubes (MWCNTs) composite films was reported for the first time. The entrapment of AO in PEDOT/MWCNTs composite films was performed during an electrochemical polymerization process. The influence of various experimental conditions was examined for determining the optimum analytical performance. The response of the biosensor towards AA under the optimized conditions is linear from 0.05 to 20 mM with a detection limit of 15 µM (S/N = 3). The biosensor shows a response time of 20 s and a sensitivity of 23.95 mA M(-1) cm(-2). The apparent Michaelis-Menten constant (K(m)) and apparent activation energy (E(a)) are 19.5 mM and 21 kJ mol(-1), respectively. Moreover, the biosensor exhibits good anti-interferent ability, good reproducibility and remarkable storage stability.