Bioactive species, including reactive oxygen species, reactive nitrogen species, and reactive sulfur species, play important roles in physiological processes; thus, it is very valuable to monitor such species in subcellular systems. Herein, for the first time, we report a turn-on highly red-emissive near-infrared (NIR) fluorescent probe, LF3 (3), for nitroxyl (HNO) which is composed of a triphenylphosphine moiety and possesses long-wavelength emission (740−840 nm), to the best of our knowledge. The probe, LF3, is highly selective and sensitive for HNO with a response time of 6 min and a detection limit as low as 15 nM. The quantum yield values LF3 and HNO added LF3 obtained from the fluorescence studies are 0.005 and 0.264, respectively. More importantly, LF3 is able to image both exogenous and endogenous nitroxyl in living cells without any interference from the background fluorescence.