Hydrogen sulfide (H2S) is an endogenous gasotransmitter and plays intriguing biological roles. To study the biological role of H2S, efficient fluorescent probes are in great demand. For imaging of H2S in deep-tissue, a two-photon probe that emits in the red wavelength region is of choice to avoid the autofluorescence from intrinsic biomolecules. Here, we disclose such a probe, which, developed based on an acetyl benzocoumarin fluorophore, can be excited at 900 nm under two-photon excitation and emit in the red region. The probe shows high reactivity, selectivity, and sensitivity in in vitro assays. Two-photon microscopic imaging of H2S in HeLa cells aided by the probe demonstrates that it is potentially useful to study H2S level changes in cells and tissues influenced by external stimuli. (C) 2017 Elsevier Ltd. All rights reserved.