Herein we report a highly selective thiol-reactive fluorescent probe based on a nucleophilic substitution reaction for detecting thiols over other relevant biological species. The probe, triphenylamine as a fluorophore and 2,4-dinitrobenzenesulfonyl chloride as a nucleophilic substitution group was synthesized and characterized. The ability to specially recognizing thiol was investigated by UV–vis and fluorescence spectrometers. Among the tested amino acids, only Cys, Hcy and GSH could turn on the fluorescence emission. It suggested that the system was a highly selective sensor for thiols. More importantly, the compound features a good linearity range and the detection limit is as low as 10⁻⁸ mol/L. In addition, as a typical biological thiol, the ability of probe to detecting Cys in living cells (HepG2 cells) via an enhancement of the fluorescence was proved.