Hypoxia is an important feature in the development of solid tumors. Nitroreductase (NTR) is closely related to the level of hypoxia, thus NTR-responsive fluorescent probes with high selectivity and sensitivity will help to evaluate the degree of hypoxia and guide personalized treatment. In this study, a near-infrared (NIR) NTR-activated composite nanoprobe 780-pNBC@MP-B was developed for tumor hypoxia imaging. The nanoprobe has a mesoporous organosilicon nanostructure functionalized with PEG for in vivo long circulation, and encapsulates a NIR molecular probe 780-pNBC for NTR imaging, which can exhibit responsive degradation to glutathione (GSH) attributed to the disulfide bond in the nanocarrier structure. Benefiting from the hydrophilic nanocarrier, 780-pNBC@MP-B with good water dispersibility and photostability was successfully applied to visualize NTR in hypoxic cells and tumor-bearing mice. Therefore, this work provides a new tool for tumor hypoxia detection and expands the new application of mesoporous silica in the field of enzyme probes.