Molecular diffusion and leakage impede the long-term retention of probes/drugs and may cause potential adverse effects in theranostic fields. Spatiotemporally manipulating the organelle-immobilization behavior of probes/drugs for prolonged tumor retention is indispensable to achieving effective cancer diagnosis and therapy. Herein, we propose a rational strategy that could realize near-infrared light-activated ribonucleic acids (RNAs) cross-linking for prolonged tumor retention and simultaneously endogenous hydrogen sulfide (H2S) monitoring in colorectal tumors. Profiting from efficient singlet oxygen (1O2) generation from Cy796 under 808 nm light irradiation, the 1O2-animated furan moiety in Cy796 could covalently cross-link with cytoplasmic RNAs via a cycloaddition reaction and realize organelle immobilization. Subsequently, specific thiolysis of Cy796 assisted with H2S resulted in homologous product Cy644 with reduced 1O2 generation yields and enhanced absolute fluorescence quantum yields (from 7.42 to 27.70%) with blue-shifted absorption and emission, which avoided the molecular oxidation fluorescence quenching effect mediated by 1O2 and validated fluorescence imaging. Furthermore, studies have demonstrated that our proposed strategy possessed adequate capacity for fluorescence imaging and endogenous H2S detection in HCT116 cells, particularly accumulated at the tumor sites, and retained long-term imaging with excellent biocompatibility. The turn-on fluorescence mode and turn-off 1O2 generation efficiency in our strategy successfully realized a diminished fluorescence cross-talk and oxidation quenching effect. It is adequately envisioned that our proposed strategy for monitoring biomarkers and prolonged tumor retention will contribute tremendous dedication in the clinical, diagnostic, and therapeutic fields.