A excited-state intramolecular proton transfer (ESIPT) fluorescent turn-on probe (FLA-1) for the detection of nitroxyl (HNO) is designed and synthesized. It consists of a flavone derivative moiety as the fluorophore, linked via an ester moiety to a diphenylphosphinobenzoyl group as a recognition receptor, which forms an aza-ylide upon reaction with HNO. Intramolecular attack of the aza-ylide on the ester carbonyl group releases a fluorescent flavone derivative. In addition, the results showed that FLA-1 exhibited a selective response to HNO over other biological reductants and can quantitatively detect HNO in the range of 0–20μM with the detection limit of 1.28×10−7M in aqueous solution. Importantly, FLA-1 was successfully applied to detect HNO in the range of 2–20μM with the detection limit of 3.96×10−7M in complex biological samples (serum) demonstrating its value in practical application.