A novel interface embedded in situ gold nanoparticles (GNPs) in chitosan hydrogel was constructed by One-step electrochemical deposition in solution containing tetrachloroauric (III) acid and chitosan. This deposited interface possessed excellent biocompatibility and good stability by characterization of attenuated total reflection Fourier-transform infrared spectra (ATR-FTIR), atomic force microscopy (AFM) and UV-vis spectra. The immobilized AChE, as a model, showed excellent activity to its substrate and provided a quantitative measurement of organophosphate pesticides involved in the inhibition action. Operational parameters, including the deposition time, tetrachloroauric (III) acid concentration have been optimized. Under the optimal electrodeposition, an amperometric sensor for the fast determination of malathion and monocrotophos, respectively was developed with detection limit of 0.001 mu g mL(-1). The simple method showed good fabrication reproducibility and acceptable stability, which provided a new avenue for electrochemical biosensor design. (c) 2007 Elsevier B.V. All rights reserved.