In this work, a highly sensitive acetylcholinesterase (AChE) inhibition-based amperometric biosensor has been developed. Firstly, a glassy carbon electrode (GCE) was modified with chitosan (Chits). Then, hollow gold nanospheres (HGNs) were absorbed onto the surface of chitosan based on the strong affinity through electrostatic adsorption. After that, L-cysteine (L-cys) was assembled on HGNs through Au-S bond. The hollow gold nanospheres were prepared by using Co nanoparticles as sacrificial templates and characterized by scanning electron microscopy, transmission electron microscopy and ultraviolet spectra, respectively. Finally, AChE was immobilized with covalent binding via -COOH groups of L-cysteine onto the modified GCE. The AChE biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy methods with the use of ferricyanide as an electrochemical redox indicator. Under optimum conditions, the inhibition rates of pesticides were proportional to their concentrations in the range of 0.1-150 and 0.1-200 μg L(-1) for chlorpyrifos and carbofuran, respectively, the detection limits were 0.06 μg L(-1) for chlorpyrifos and 0.08 μg L(-1) for carbofuran. Moreover, the biosensor exhibited a good stability and reproducibility and was suitable for trace detection of pesticide residues in vegetables and fruits.