A dual-activatable, fluorogenic probe was developed to sense esterase activity with single-mol. resoln. Without enzymic preactivation, the diazoindanone-based probe has an electron-poor core and, upon irradn., undergoes Wolff rearrangement to give a ring-expanded xanthene core that is nonemissive. If the probe is preactivated by carboxylesterases, the tricyclic core becomes electron-rich, and the photoinduced Wolff rearrangement produces a highly emissive rhodol dye. Live-cell and soln. studies confirmed the selectivity of the probe and revealed that the photoactivated dye does not diffuse away from the original location of activation because the intermediate ketene forms a covalent bond with surrounding macromols. Single-mol. localization microscopy was used to reconstruct a super-resolved image of esterase activity. These single-mol. images of enzymic activity changed significantly upon treatment of the cells with inhibitors of human carboxylesterase I and II, both in terms of total no. of signals and intracellular distribution. This proof-of-principle study introduces a sensing mechanism for single-mol. detection of enzymic activity that could be applied to many other biol. relevant targets.