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A lateral field excited AlN film bulk acoustic enzyme biosensor was reported for the highly sensitive detection of residual pesticides. Acetylcholinesterase enzyme is assembled on one of the faces of the piezoelectric resonator as the sensitive coating. The enzymatic reaction in the substrate solution and the inhibitory effect of organophosphorus compounds on the enzymatic activity are observed by measuring the frequency shift in situ and in real time. The traces of organophosphorus pesticides in the solution can be detected from the diminution of the frequency shift compared with the levels found in their absence. The device exhibits linear responses, simple operation, portability, and a remarkable detection limit of 4.3 × 10-11M for ethyl para-nitro-phenyl (a typical organophosphorous pesticide). The detection results of residual pesticides in practical samples show that the proposed biosensor has the feasibility and accuracy comparable to gas chromatography.