Acetylcholinesterase (AChE) is popularly used in biological and chemical sensors since it can be inhibited or reactivated. As the inhibitor of AChE is meaningful for many neurodegenerative diseases caused by the lack of AChE substrate acetylcholine (ACh), and the reactivator of inhibited AChE is helpful to treat the blocked neurotransmission resulted from the excessive ACh. Therefore, it is significant to screen AChE inhibitors and reactivators for pharmaceutical application. In addition, the detection of pesticides and heavy metals is of great importance to food safety and environment. So, based on the inhibition of AChE by pesticides and heavy metals, AChE can also be used in food safety and environmental analysis. Colorimetric method is a popular strategy for AChE assay due to its low cost, practicality and simplicity. In this review, we focus on the colorimetric method for AChE assay and the corresponding applications. The basic principles, advantages and disadvantages of Ellman's method, pH indicator-, 3,3′,5,5′-tetramethylbenzidine- (TMB-), noble metal nanomaterial-, and substrate-based colorimetric method are evaluated. In addition, the applications of colorimetric AChE assays in inhibitors and reactivators screening, pesticides and heavy metals detection are reviewed. Finally, the conclusions and future perspectives are provided. Display omitted •Different chromogenic reagent-based colorimetric method for AChE are reviewed.•The mechanism, advantage and disadvantage of colorimetric AChE assay are discussed.•The applications of colorimetric AChE assays in analysis are reviewed.•A perspective for the colorimetric AChE assay is proposed.