Nitroreductase (NTR) is an endogenous reductase overexpressed in hypoxic tumors, with its levels closely correlated to the degree of hypoxia. This correlation has significant clinical implications for the analysis of tumor hypoxia, as it allows for the indirect detection of nitroreductases. Due to their simplicity, noninvasive nature, and excellent spatiotemporal resolution, various fluorescence methods have been developed for the analysis of nitroreductase and tumor hypoxia. In this study, we present the design, synthesis, in vitro evaluation, and biological application of an NTR-activated fluorescent probe, F-NTR. Utilizing an oxanthrene fluorophore as the core component, F-NTR incorporates a 4-nitrobenzene recognition group. This innovative probe, which introduces a nitro group, demonstrates high selectivity and reactivity towards nitroreductase (NTR) due to its reducing properties. Furthermore, probe F-NTR is capable of accurately identifying hypoxic environments, which provides a basis for precise detection and localization of tumors. This work lays the groundwork for future investigations into cell metabolism, tumor metabolism, and the surgical management of solid tumors under hypoxic conditions.