Direct visualization of Nitroxyl (HNO) release is currently not feasible for all chemical and biological systems, limiting the understanding of its biological and pharmaceutical activities. To address this issue, N-hydroxydansylamide was developed as the first fluorescence turn-off HNO donor that efficiently releases HNO with a fluorescence turn-on probe. Under physiological conditions, the HNO release was observed with a half-life of 19.86 min. The decomposition of N-hydroxydansylamide yielded a fluorescent dansyl-based sulfinate species. Specifically, the concentration of HNO released was successfully monitored based on the fluorescence signal with a limit of detection of ~ 0.55 µM. Particularly, HNO releasing can be sensed without any depletion of the HNO source. Finally, cellular imaging was also achieved to demonstrate the HNO release derived from N-hydroxydansylamide. Therefore, HNO release profiles in terms of kinetics, dose, and the site can be monitored through real-time fluorometric analysis or imaging-based studies, providing a novel platform to explore HNO biology.