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Fluorescence detection of H 2 S in living organisms is greatly advantageous because it is nondestructive and can be used for in situ analysis. We have constructed a novel rhodamine analogue dye (Rho630) by extending the conjugated system of rhodamine to create a novel cell-trappable H 2 S fluorescent probe Rho630-AM-H 2 S with red light emission. Its application for H 2 S fluorescence detection in living HeLa cells and zebrafish was investigated. As expected, Rho630-AM-H 2 S showed a huge fluorescence turn-on response of about 20-fold at 630 nm and good selectivity toward H 2 S in solution. An MTT assay demonstrated that the probe showed negligible cytotoxicity in the concentrations typically used in fluorescence imaging experiments. Cell imaging experiments revealed that compared with compound 4 without cell-trappable unit modification, Rho630-AM-H 2 S exhibited remarkably enhanced cell penetration ability, as an enormous fluorescence signal increase was observed at the red channel within 5 min after Rho630-AM-H 2 S was incubated with HeLa cells. Finally, the probe Rho630-AM-H 2 S was used to detect H 2 S in living HeLa cells and zebrafish with great fluorescence enhancement in the red channel. Graphical abstract.