Tumor microenvironment response and spatio-temporal targeting are promising strategies to combination therapy. By regulating the tumor microenvironment and realizing the precise targeted therapy, the antitumor efficacy can be significantly improved while maintaining low side effects. Therefore, in this study, a reactive oxygen species (ROS)-responsive nanosystem (MND-IR@RESV) was constructed. The MND-IR@RESV nanosystem could be accurately tracked and located due to its own fluorescence. Mitochondria-targeted nanosystem promoted the release of ROS and contributed to the apoptosis of cancer cells. The MND-IR@RESV responded significantly to ROS, which promoted the deep release of drugs. Furthermore, the system also possessed high photothermal conversion efficiency and photothermal stability, which could achieve spatio-temporal targeting of drugs and ensure the efficient tumor eradication ability of Chemo/Photothermal/Photodynamic (CT-PTT-PDT) combination therapy. Both in vitro and in vivo tests demonstrated the significant antitumor effect of the MND-IR@RESV. Importantly, the nanosystem had the advantages of low toxicity and few side effects that conventional CT lacks, indicating that the MND-IR@RESV holds great promise in future cancer treatment.