Acetylcholinesterase (AChE) plays an essential role in biological signal transmission, the aberrant expression of which could cause diverse neurodegenerative diseases. Herein, based on the oxidase-like activity of manganese dioxide nanosheets (MnO NSs), we found that MnO NSs could directly oxidize thiamine into intensely fluorescent thiochrome without the need of peroxides. When AChE was introduced, acetylthiocholine could be hydrolyzed to generate thiocholine, which efficiently triggered the reduction of MnO NSs into Mn, resulting in the decrease of fluorescence. Owing to the inhibiting effect of tacrine to the AChE activity, the decomposition of MnO was hindered, thus leading to the fluorescence recovery. According to the above mechanism, we constructed a simple, low-cost, label-free, facile and rapid synthetic fluorescent biosensor for highly sensitive and selective detection of AChE activity and screening of its inhibitor. This biosensor obtained a good linear range from 0.02 to 1 mU/mL and an extremely low detection limit of 15 μU/mL for AChE assay, as well as a sensitive screening for tacrine and an excellent applicability in human serum samples. These results suggested that our proposed method would be potentially applied in monitoring the disease progression.