Hydrogen sulfide (H2S) plays crucial roles in antioxidation, anti-inflammation, and cytoprotection. Despite the great progress in the design and synthesis of activatable H2S donors, high-precision detection and imaging of released H2S in living systems is still an outstanding issue. In this study, a biothiol-activated near-infrared (NIR) fluorescent H2S donor PRO-ST for real-time visualization of H2S release is developed. PRO-ST, consisting of a dicyanoisophorone-based NIR fluorescence moiety (TCOO), a biothiol-trigger moiety (4-isothiocyanate benzyl alcohol), and a sulfur-source group (thiophosgene), is characterized by large NIR fluorescence enhancement (45-fold), outstanding H2S release efficiency (73%), prominent controllable H2S release (60 min) and excellent cell compatibility. The distinctive features render PRO-ST capable of visualizing H2S release in cells, zebrafish, and mice. Moreover, PRO-ST has exhibited excellent performance of real-time anti-inflammation and wound healing enhancement in biological systems, which has been confirmed by in-situ visualization of H2S release. The versatile PRO-ST provides possible and effective solution to the detection and visualization of H2S release, which will benefit the comprehension of underlying mechanisms of wound healing and the optimization of interventional therapy.