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Hydrogen sulfide (H2S) is a critical gasotransmitter playing an important role in various physiological and pathological processes. The development of fluorescent probes for rapid, sensitive, and selective lysosomal H2S sensing has attracted much attention. Herein we report a rhodamine based probe MF‐N3 that can selectively accumulate in lysosomes and give turn‐on fluorescence in the presence of both H2S and protons. Results show that MF‐N3 has an exclusive response to H2S in the acidic lysosomal environment but does not respond in other organelles or in neutral environments. This strategy can circumvent the fluorescence interference from H2S of other sources, thus providing a high sensitivity and selectivity on lysosomal H2S imaging by fluorescence microscopy. This pH‐dependent lysosome‐targeting fluorescent probe has been successfully applied to lysosomal H2S imaging in living cells and is of great potential for applications to the environmental and biological systems. [ABSTRACT FROM AUTHOR]