The abnormal expression of acetylcholinesterase (AChE) is linked to the development of various diseases. Accurate determination of AChE activity as well as screening AChE inhibitors (AChEIs) holds paramount importance for early diagnosis and treatment of AChE-related diseases. Herein, a fluorescent and colorimetric dual-channel probe based on gold nanoclusters (AuNCs) and manganese dioxide nanosheets (MnO₂ NSs) was developed. The fluorescence of AuNCs was suppressed in the presence of MnO₂ NSs, providing a platform for fluorescence-based detection. For colorimetric, the nanocomposites exhibited oxidase-like activity, rapidly catalyzing 3,3′,5,5′-tetramethylbenzidine (TMB) to generate a blue color. Thiocholine (TCh), produced through the enzymatic reaction of acetylthiocholine (ATCh) in the presence of AChE, can reduce the MnO₂ NSs, thus recovering the suppressed fluorescence of AuNCs and decreasing oxidase-like activity. Based on this principle, a dual-mode assay for AChE detection was achieved. Compared to the commonly used single-signal detection, multi-mode detection can offer reliable and accurate results, due to its inherent self-validation and self-regulation capabilities. Under optimum conditions, the limit of detection (LOD) for AChE activity was 0.067 mU mL^–1 (fluorescent mode) and 0.042 mU mL^–1 (colorimetric mode). Moreover, the probe realized AChE detection in biological samples and AChEIs screening. This work showed the great prospects for early diagnosis of AChE-related diseases. Graphical Abstract