We have presented a near-infrared fluorescent probe Lyso-JN for the detection of nitroxyl (HNO) in cells and in vivo. Lyso-JN is comprised of three moieties: an Aza-BODIPY fluorophore, a HNO-response modulator, diphenylphosphino-benzoyl, and a lysosomal locator, alkylmorpholine. The detection mechanism is based on aza-ylide intramolecular ester aminolysis reaction with HNO. The probe holds the ability to capture lysosomal HNO in RAW 264.7 cells, and it is also successfully employed to visualize HNO in mice.