Glutathione (GSH) is known to play a key role in the modulation of the redox environment in N-methyl-d-aspartate (NMDA) receptors. Coumarin derivative 1 bearing cyanoacrylamide and ifenprodil moieties was synthesized and reported to monitor GSH near NMDA receptors. The cyanoacrylamide moiety allows probe 1 to monitor GSH reversibly at pH 7.4 and the ifenprodil group acts as a directing group for NMDA receptors. Two-photon fluorescence microscopy allows probe 1 to successfully sense endogenous GSH in neuronal cells and hippocampal tissues with excitation at 750 nm. Furthermore, the addition of H2O2 and GSH induced a decrease and an increase in fluorescence emission. Probe 1 can serve as a potential practical imaging tool to get important information on GSH in the brain.