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Glutathione (GSH) plays crucial roles in various physiological and pathological processes. The fluctuation of the GSH level is closely associated with a variety of diseases and cellular functions. Hence, it is important to real-time monitor the fluctuation of GSH in living cells. In this work, we presented a rhodol-hybridized hemicyanine fluorophore (RdH) as a selective, rapid-response, ratiometric, and reversible fluorescent probe for intracellular GSH (t1/2 = 89 ms, Kd = 1.42 mM). The imaging assays in living cells revealed that RdH could be used to real-time monitor GSH dynamics in A549 cells under a laser scanning confocal microscope by ratiometric fluorescence changes. [ABSTRACT FROM AUTHOR]