An amperometric biosensor for the detection of organophosphorus pesticide was developed based on layer-by-layer self-assembly both acetylcholinesterase(AChE) and gold nanoparticles(AuNPs) on glassy carbon electrodes（GCE) in the presence of poly(diallyldimethylammonium chloride)（PDDA).The electrochemical properties of the biosensor were investigated by electrochemical impedance spectroscopy and differential pulse voltammetry.The proposed biosensor exhibited a high affinity to the substrate of AChE and produced a detectable and fast response owing to its excellent electron transfer rate and satisfactory biocompatibility of AuNPs.Compared with that at the unmodified electrode,the peak current of thiocholine at the proposed biosensor obviously increased.Under the optimum conditions,the inhibition rate of methyl parathion to the immobilized AChE was proportional to the logarithm of concentration of methyl parathion over the range of 4.6×10-5 – 5.3×10-3 mol/L with a detection limit of 7.6×10-6 mol/L.The proposed biosensor was successfully applied in the determination of methyl parathion in vegetable sample,which means that the proposed biosensor provides a new promising tool for the pesticide analysis.