Glutathione (GSH) plays many important roles in maintaining intracellular redox homeostasis, and determining its real-time levels in the biological system is essential for the diagnosis, treatment, and pathological research of related diseases. Fluorescence imaging has been regarded as a powerful tool for tracking biomarkers in vivo, for which specificity, reversibility, and fast response are the main issues to ensure the real-time effective detection of analytes. The determination of GSH is often interfered with by other active sulfur species. However, in addition to the common features of nucleophilic addition, GSH is unique in its large molecular scale. 2-(2-Hydroxyphenyl) benzothiazole (HBT) was often formed in the ESIPT process. In this study, HBT was installed with α,β-unsaturated ketone conjugated coumarin derivates or nitrobenzene, which were used to adjust the reactivity of α,β-unsaturated ketone. Experimental and theoretical calculations found ESIPT to be favorable in HBT-COU but not HBT-COU-NEt2 or HBT-BEN-NO2 due to the higher electronic energies in the keto form. Thus, for HBT-COU, in the presence of GSH, the hydrogen-bonding interaction between C═N of the HBT unit and carboxyl of GSH would inhibit the process, simultaneously promoting the Michel addition reaction between α,β-unsaturated ketone and GSH. As a consequence, probe HBT-COU could exhibit a rapid reversible ratiometric response to GSH. Small structures of Hcy and Cys are passivated for such reactions. Cell imaging demonstrated the specific response of the probe to GSH, and the probe was successfully used to monitor fluctuations in GSH concentration during cells apoptosis in real-time.