Single-atom nanozymes (SAzymes), with well-defined and uniform atomic structures, are an emerging type of natural enzyme mimics. Currently, it is important but challenging to rationally design high-performance SAzymes and deeply reveal the interaction mechanism between SAzymes and substrate molecules. Herein, we reported the controllable fabrication of a unique Cu-N 1 S 2 -centred SAzyme (Cu-N/S-C) via a chemical vapor deposition-based sulfur-engineering strategy. Benefiting from the optimized geometric and electronic structures of single-atom sites, Cu-N/S-C SAzyme shows boosted enzyme-like activity, especially in catalase-like activity, with a 13.8-fold increase in the affinity to H 2 O 2 substrate and a 65.2-fold increase in the catalytic efficiency when compared to Cu-N-C SAzyme with Cu-N 3 sites. Further theoretical studies reveal that the increased electron density around single-atom Cu is achieved through electron redistribution, and the efficient charge transfer between Cu-N/S-C and H 2 O 2 is demonstrated to be more beneficial for the adsorption and activation of H 2 O 2 . The as-designed Cu-N/S-C SAzyme possesses an excellent antitumor effect through the synergy of catalytic therapy and oxygen-dependent phototherapy. This study provides a strategy for the rational design of SAzymes, and the proposed electron redistribution and charge transfer mechanism will help to understand the coordination environment effect of single-atom metal sites on H 2 O 2 -mediated enzyme-like catalytic processes. This article is protected by copyright. All rights reserved.