An NBD-based fluorescent probe (probe 1) for the detection of Hcy over Cys and GSH based on nucleophilic substitution and Smiles rearrangement reaction was designed and synthesized by the reaction of 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) with 1,4-dihydroxybenzene. The probe displayed many desired properties such as high specificity, appreciable solubility, and desirable response time. In addition, probe 1 has low cytotoxicity and detection of intracellular Hcy can be achieved by cell fluorescence imaging.