Glutathione (GSH), the most abundant nonprotein thiol found in living organisms, are involved in the etiology and progression of many human diseases including cancer. So, monitoring changes of cellular GSH levels has an important guiding significance. To date, however, majority of probes can only qualitatively detect GSH in living cells. Herein, with coumarin as the read-out fluorophore and Michael addition as the sensing mechanism, six fluorescent probes were designed and synthesized. Among them, RP-2 exhibited a reversible and extremely fast response toward GSH (half time: ∼3 s), which endowed RP-2 the capacity of real-time imaging. Among the reversible probes based on Michael addition, RP-2 had both the largest forward and reverse rate constants thus far. The reaction between RP-2 and GSH was studied in detail by density functional theory and fluorescence spectroscopy. Real-time imaging of GSH levels in living cells was achieved with a temporal resolution of seconds. To simplify the processing of images, a program was developed and validated. RP-2 was expected to serve as a new fluorescent imaging tool to understand the function of intracellular GSH in the future.