<p>As the unique tumor microenvironment is indispensable for cancer cell survival, disrupting redox homeostasis in tumor cells was a promising strategy for effective tumor treatment. Herein, a novel vanadium-doped carbon dots (V-CDs) nanozyme was prepared via a hydrothermal method. Through appropriate selection of precursors, V-CDs with mixed valence states (V⁴⁺ and V⁵⁺) were obtained. It was the unique vanadium doping that endowed the as-prepared V-CDs with remarkable fluorescence performance and multi-enzyme-like activities, including oxidase (OXD)-like, peroxidase (POD)-like, and glutathione peroxidase (GPx)-like catalytic behaviors. Based on the above properties of V-CDs, their applications in colorimetric sensing and anticancer activity had been fully explored. A simple colorimetric sensing for L-Cysteine (L-Cys) was established based on the 3,3’,5,5’-tetramethylbenzidine&#xa0;(TMB) color reaction. Moreover, V⁴⁺ generated hydroxyl radicals (<b>·</b>OH) and singlet oxygen (¹O₂) from hydrogen peroxide (H₂O₂) through Fenton-like reactions and the Russell mechanism, while V⁵⁺ within V-CDs could effectively deplete GSH. V-CDs exhibited anticancer effect, which likely results from the synergistic actions of multi-enzyme-like activity. The generation of reactive oxygen species (ROS), combined with the depletion of overexpressed glutathione (GSH), demonstrated considerable inhibitory effect on cancer cells proliferation.</p> Graphical Abstract <p></p>