Hydrogen sulfide (H2S) as a key fundamental gasotransmitter regulates various biological processes, and the incontrollable H2S is essentially associated with the occurrence and development of multiple diseases, including cancers. Photodynamic therapy (PDT), as an invasive tumor treatment technology, has also attracted great attentions. Due to the key role of elevated H2S in cancers, integrating H2S depletion/recognition and PDT should be an effective strategy to enhance anticancer performance. In this work, we report a H2S depletion aided PDT platform (3RAX-NBD) by the chemical ligation of 3RAX and NBD. 3RAX-NBD can react rapidly with H2S and generate a novel 3RAX derivative compound 3 with increased fluorescence in vitro and in vivo. More notably, 3RAX-NBD can effectively kill multiple cancer cells through in situ irradiation, and 3RAX-NBD also has prominent anticancer effects on 4 T1 tumor-bearing BALB/c female mice with no notably toxic side effects. We believe that our H2S depletion aided PDT platform may provide a powerful tool for studying the key roles of H2S in diseases, and also give another promising candidate for cancer treatment. A novel PDT platform (3RAX-NBD) was developed with dual functions of H2S recognition and photodynamic activity which has prominent therapeutic effects and biosafety in vivo. Display omitted •Design and synthesis a novel compound (3RAX-NBD) with dual functions of H2S recognition and photodynamic activity.•3RAX-NBD has a specific response to H2S with a low detection limit, and can be used for imaging exogenous H2S in living cells.