Single-atom nanozymes (SAzymes), as novel nanozymes with atomically dispersed active sites, are of great importance in the development of nanozymes for their high catalytic activities, the maximum utilization efficiency of metal atoms, and the simple model of active sites. Herein, the peroxidase-like SAzymes with high-concentration Cu sites on carbon nanosheets (Cu–N–C) were synthesized through a salt-template strategy. With the densely distributed active Cu atoms (∼5.1 wt %), the Cu–N–C SAzymes exhibit remarkable activity to mimic natural peroxidase. Integrating Cu–N–C SAzymes with natural acetylcholinesterase and choline oxidase, three-enzyme-based cascade reaction system was constructed for the colorimetric detection of acetylcholine and organophosphorus pesticides. This work not only provides a strategy to synthesize SAzymes with abundant active sites but also gives some new insights for robust nanozyme biosensing systems.