The invention relates to an acetylcholinesterase biosensor and an application thereof, and belongs to the technical field of electrochemical detection. The biosensor is produced through the following steps: 1, preparing a MnO2-nanoflakes composite nano-material and a Pd-Pt@MnO2-nanoflakes composite nano-material from KMnO4, CTAB, PdCl2, PEG400, H2PtCl6, sodium citrate and sodium borohydride used as initial raw materials through a water bath technology; and 2, immobilizing acetylcholinesterase by using chitosan (CS), and immobilizing acetylcholinesterase (AChE) on the surface of a Pd-Pt@MnO2 modified glassy carbon electrode through adopting physical adsorption to construct the electrochemical biosensor. The biosensor adopting an electrochemistry and enzyme sensing combination technology realizes high-sensitivity detection of methyl parathion and carbofuran pesticides, has the advantages of small amount of a required sample, short detection time, high sensitivity and low cost, and is suitable for analyzing and detecting pesticide residual.