Hydrogen polysulfides (H2Sn, n > 1), derived from hydrogen sulfide (H2S), have attracted increasing attention in biochemical research, which may perform as the actual signaling molecules during cell signaling processes. Because of the closed biological and chemical relationship between H2S and H2Sn, it is of great value to develop sensitive and specific techniques to distinguish the intracellular level of H2Sn. To improve the understanding of the physiological and pathological roles played by H2Sn, we now develop a specific fluorescent probe Mito-ss for capturing H2Sn in cells and in vivo. When triggered by H2Sn, Mito-ss replies a turn-on fluorescence signal and exhibits a higher selectivity toward H2Sn than other abundant competing biothiols, such as glutathione, cysteine and H2S. The probe Mito-ss can also be applied to visual H2Sn in living cells, as well as in vivo, providing a potentially powerful approach for probing H2Sn in biological systems.