Nitroreductase (NTR), one of the flavin-dependent enzymes and an upregulated enzyme under tumor hypoxia, has been studied for decades. Many fluorescent probes were developed to detect NTR activity; however, these probes tend to diffuse away from their reaction site (NTR) inevitably, leading to inappropriate sample fixation, lower accuracy of NTR localization, and weaker signal-to-noise ratio. Herein, we present the design, synthesis, in vitro evaluation, and biological applications of an NTR-activatable fluorogenic and labeling probe FY. By integrating with quinone methide (QM) proximity-based protein labeling, the additional fluoromethyl group on FY serves as a potential origin of QM. Compared with conventional fluorescent probes, this new NTR probe not only offers mitochondrial localizable and fluorogenic response but also achieves permanent retention on the site of activation with an enhanced spatial resolution to improve the detection sensitivity even after cell fixation. We believe our work could offer an expandable synthetic approach to develop these permanent labeling and imaging fluorescence probes for deciphering complex biological events.