The detection of center dot OH in live organisms is crucial to the understanding of its physiological and pathological roles; while this is too challenging because of the extremely low concentration and high reactivity of the species in the body. Herein, we report the rational design and fabrication of an NIR-light excited luminescence resonance energy transfer-based nanoprobe, which for the first time realizes the in vivo detection of center dot OH. The nanoprobe is composed of two moieties: upconversion nanoparticles with sandwich structure and bared surface as the energy donor; and mOG, a modified azo dye with tunable light absorption, as both the energy acceptor and the center dot OH recognizing ligand. The as-constructed nanoprobe exhibited ultrahigh sensitivity (with the quantification limit down to 1.2 femtomolar, several orders of magnitude lower than that of most previous center dot OH probes), good biocompatibility, and specificity. It was successfully used for monitoring [center dot OH] levels in live cells and tissues.