Acetylcholinesterase (AChE) is the key enzyme in the treatment of Alzheimer's disease (AD). In this study, we designed a novel visualization biosensing system for the quantitative detection of AChE and its inhibitors. The MnO2–OPD platform showed the broad detection of AChE ranging from 0.49–98 U L−1 and the LOD was 0.13 U L−1. Furthermore, the AChE–MnO2–OPD biosensing system was successfully applied to screen AChE inhibitors from a natural product library (Northwestern China Plant Natural Material Bank). 3,4-Dihydroxyphenylethanol (HJ-38), a natural product isolated from Zanthoxylum bungeanum pericarps, was found to be a potent AChE inhibitor with an IC50 of 2.8 μM and is comparable to the positive control tacrine with an IC50 of 1.3 μM. Furthermore, a cell-based assay confirmed the neuro-protective potential of compound HJ-38, which supported the practicability of the established AChE–MnO2–OPD biosensing system.