To explore highly sensitive and low‐toxicity techniques for tracking and evaluation of non‐small‐cell lung cancer (NSCLC), one of the most mortal tumors in the world, it is utterly imperative for doctors to select the appropriate treatment strategies. Herein, developing near‐infrared (NIR) excited nanosensors, in which the donor and acceptor pairs within a biological metal–organic framework (bio‐MOF) matrix are precisely controlled to rationalize upconversion Förster resonance energy transfer (FRET), is suggested for detecting the O2 concentration inside tumors with reduced signal disturbance and health detriment. Under NIR excitation, as‐fabricated core/satellite nanosensors exhibit much improved FRET efficiency and reversible hypoxic response with high sensitivity, which are effective both in vitro and in vivo (zebrafish) for cycling normoxia–hypoxia imaging. Significantly, combined with a reliable preclinical genetically engineered murine model, such nanosensors successfully realize tracking of in vivo NSCLC lesions upon clear and gradient hypoxia signals without apparent long‐term biotoxicity, illustrating their exciting potential for efficient NSCLC evaluation and prognosis.