Nitroxyl (HNO), the one-electron reduced and protonated analogue of nitric oxide (NO), demonstrates distinctive bio-pharmacological effects in the treatment of cardiovascular disorders. Herein, we design and synthesize a near-infrared (NIR) metal-free fluorescent probe Cyto-JN for the detection of nitroxyl (HNO) in living cells and in vivo. The metal-free Cyto-JN is composed of two moieties: the Aza-BODIPY fluorophore and the HNO recognition unit, the diphenylphosphinobenzoyl group. Cyto-JN can react with HNO in a 1 : 1 stoichiometry, which may bring great benefit to the detection efficiency of bioassays. Cyto-JN shows high sensitivity toward HNO and exhibits low cytotoxic effect on cells. Moreover, the probe displays good selectivity for the detection of HNO in the presence of various biologically related species. Cyto-JN can be applied successfully to bio-imaging of HNO in living cells and in mice. The results of flow cytometry confirm that the probe Cyto-JN can be used to detect intracellular HNO qualitatively and quantitatively.