Developing activatable photodynamic agents is becoming a promising mode for realizing selective photodynamic therapy (PDT) in cancer treatment. However, till now only a few H2S-activatable photodynamic agents have been involved in this field. Here, we fabricated H2S-activatable nano-assemblies (P@TPPCY) for the treatment of colon cancer containing high concentration H2S. The H2S-activatable photosensitizer (TPPCY) was synthesized by covalent conjugation between tetraphenylporphyrin (TPP) and heptamethine cyanine (Cy7), and then TPPCY was encapsulated by an amphiphilic polymer DSPE-mPEG to form P@TPPCY nano-assemblies. We demonstrated that the photosensitizing effect of TPP was efficiently quenched by Cy7 with strong absorption in the NIR region via fluorescence resonance energy transfer (FRET) effect. Interestingly, the FRET effect between Cy7 and TPP was attenuated by the decrease of absorption of Cy7 in the NIR region after the Cy7 reacted with H2S, and then the photosensitizing effect of TPP in P@TPPCY was activated. Strikingly, the P@TPPCY showed efficient H2S-activatable photodynamic therapy (PDT) in vitro against HCT116 cells (human colon carcinoma cell line), thus this work provides a new method for realizing accurate treatment of colon cancer in virtue of high H2S concentration microenvironment.