Abnormal hypoxic stress triggers a cascade of pathological consequences, particularly in tumorigenesis. A comprehensive understanding of hypoxia's role in tumor development is crucial for creating effective therapies. This requires reliable tools that can specifically identify hypoxic tumor cells (or tissues) and correlate their dynamics with disease progression in complex living environments, enabling precise theranostics. Nevertheless, a key limitation is the tendency of these probes to disperse from their intended enzymatic targets, which compromises their accuracy in enzyme localization and diminishes the signal-to-noise ratio. Herein a novel ESIPT-based fluorescent probe, BTN, was developed for the detection of NTR. When exposed to NTR, BTN demonstrated a marked fluorescence off-to-on transition at 610 nm, accompanied by a detection limit as low as 0.078 μg/mL. Fluorescence imaging further confirmed its capability to identify the heightened expression of NTR in cancerous cells. Additionally, it was demonstrated that downregulation of HK2 triggered ferroptosis, thereby inhibiting the growth and invasion of lung cancer cells. More significantly, BTN can in situ monitor the inhibition of lung cancer progression by HK2 knockdown under hypoxic conditions (1 % O₂). We anticipate that this probe will provide a valuable tool for gaining a deeper understanding of the biological functions of NTR in cancer, as well as uncovering critical insights into hypoxia-related pathological processes.