7-nitro-2,1,3-benzoxadiazole (NBD)-based fluorescent probe for the detection of cysteine (Cys) over homocysteine (Hey) and amino acids under neutral physiological conditions based on thiol-induced aromatic nucleophilic substitution reaction was synthesized by the reaction of 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) with p-thiocresol. The proposed sensing mechanism was proved by mass spectrometry of the reaction mixtures. This probe was successfully applied to the fluorescence imaging of Cys in HeLa cells.