A new 4-hydroxy-1,8-naphthalimide-based ratiometric fluorescent probe for hydrogen sulfide (H 2 S) detection was designed and synthesized. The probes showed fast response, high sensitivity and selectivity for hydrogen sulfide over other reactive sulfur species. The mechanism is based on H 2 S-mediated nucleophilic addition followed by an intramolecular cyclization to give the product, 4-hydroxy-1,8-naphthalimide, which showed outstanding intramolecular charge transfer (ICT). Therefore, ratiometric signal was observed and with a H 2 S detection of 1–10μM. Furthermore, the probe successfully applied to bioimaging, demonstrating its potential applications in biological systems.