Hydrogen polysulfides (H2Sn, n > 1) are crucial for physiological functions and pathological processes. They are highly desirable for developing mitochondria-targetable fluorescent probes to monitor the fluctuation of H2Sn level when the intracellular redox state changes. Here, we report a ratiometric fluorescent probe Mito-RHP for detecting H2Sn in mitochondria by integrating the triphenylphosphonium and 2-fluoro-5-nitrobenzoic ester groups with a 1,8-naphthalimide-based fluorophore. High sensitivity and selectivity to endogenous and exogenous H2Sn was observed. Mito-RHP was stable under normal conditions and was an efficient fluorescent probe for bioimaging. Bioimaging experiments demonstrated that Mito-RHP possessed a good mitochondria-targeting property and showed a ratiometric response to H2Sn. Further, it could be used for H2Sn detection in living cells under stimulus-induced redox imbalance. These advantages enable us to further understand H2Sn-related physiological and pathological effects.