The goal of this preliminary study was to produce a promising alternative tool for the direct, rapid and specific determination of vitamin C (VC) content in real samples of different agricultural crops without pre-treatment. For this purpose, a VC electrochemical biosensor based on one-step immobilization of ascorbate oxidase in the biocompatible conducting poly(3,4-ethylenedioxythiophene)-lauroylsarcosinate (PEDOT-SL) film was facilely developed. AO molecules were incorporated into the resulting PEDOT-SL film by using a co-electrodeposition technique under optimum conditions. The sodium N-lauroylsarcosinate provided a good biocompatible environment for biologically-active macromolecules AO. The electrochemical characterization and scanning electron microscopy indicated that AO molecules were successfully immobilized in this PEDOT-SL film. The parameters of the biosensor such as the film thickness, working potential, pH, concentration of PBS, and temperature were optimized. The biosensor displayed an excellent bioelectrocatalytic activity to the oxidation of VC and could successfully detect VC in the concentration range of 0.002-14 mM. The biosensor also exhibited a high sensitivity of 80.4 mA M-1 cm(-2), a fast response time of 15 s, a low working potential of 0.2 V, a low detection limit of 0.464 mu M. In addition, high bioaffinity, bioactivity, stability and specificity of this biosensor were also evaluated. The excellent performance of the prepared biosensor and good results of the VC determination in commercial juices and vegetable crops indicated that the as-fabricated biosensor will be a good candidate for the detection and analysis of VC in agricultural crops. (C) 2012 Elsevier B.V. All rights reserved.