The emergence of carbon dots-based nanozymes (CDNEs) has opened a broad horizon for multi-mode analysis and safety antibacterial application. However, the practical applications of CDNEs are currently hindered by unsatisfactory performance and complicated synthetic methodology. In this study, we developed a novel copper-doped CDNEs (Cu-CDNEs) with both excellent fluorescence property and peroxidase-like activity. The Cu-CDNEs can be synthesized easily by pyrolyzing a solid mixture of copper glutamate and ethylenediamine hydrochloride for 5 min. By investigating the inhibition of H2S on the peroxidase-like activity of Cu-CDNEs and the competitive interaction between H2S and 3,3′,5,5′-tetramethylbenzidine with reactive oxygen species, we established a dual-mode analysis system for colorimetric and fluorescent detection of H2S with the limit of detection of 20 nM and 25 nM, respectively. Furthermore, a convenient scanometric analysis mode also was developed and successfully applied for the accurate determination of H2S in actual water samples with recoveries of 96.8 %–107.7 %. Moreover, the designed Cu-CDNEs show good cytocompatibility from MTT results, and demonstrated broad-spectrum antibacterial capacity against Gram-positive/negative bacteria due to the surface positive charge and peroxidase-like activity. This study may provide valuable insights for rational design and broad applications in environmental and biomedical fields using functional nanozymes.