Real-time tracking of GGT enzymatic activity in human ovarian cancer cells is a reliable method for accurate prediction of cancer diagnosis and management. Here, we report the two-photon ratiometric tracking of GGT activity in cancer cells based on a probe with switchable Förster resonance energy transfer properties. In the absence of GGT, the designed probe showed two well-resolved emission bands at 461 and 610 nm, corresponding to the 7-hydroxycoumarin donor and BODIPY acceptor, respectively. In contrast, GGT catalyzed cascade reactions including cleavage of the γ-glutamyl group and subsequent aromatic hydrocarbon transfer from the S to N atom increased the distance between the two chromophores, thus decreasing the FRET efficiency, with the recovery of the donor fluorescence at 461 nm. By exploiting this enzyme-triggered ratiometric measurement, successful differentiation of ovarian cancer cells from normal cells with this probe was realized by two-photon fluorescence confocal microscopy.