In this paper, chitosan-prussian blue-multiwall carbon nanotubes-hollow gold nanospheres (Chit-PB-MWNTs-HGNs) film was fabricated onto the gold electrode surface by one-step electrodeposition method; and then acetylcholinesterase (AChE) and Nafion were modified onto the film to prepare an AChE biosensor. Incorporating MWNTs and HGNs into Chit-PB hybrid film promoted electron transfer reaction, enhanced the electrochemical response and improved the microarchitecture of the electrode surface. The morphologies and electrochemistry properties of the composite were investigated by using scanning electron microscopy, transmission electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy, respectively. Parameters affecting the biosensor response such as pH, enzyme loading and inhibition time were optimized. Based on the inhibition of pesticides on the AChE activity, using malathion, chlorpyrifos, monocrotophos and carbofuran as model compounds, this biosensor showed a wide range, low detection limit, good reproducibility and high stability. Moreover, AChE/Chit-PB-MWNTs-HGNs/Au biosensor can also be used for direct analysis of practical samples, which would be a new promising tool for pesticide analysis.