Hydrogen sulfide (HS) has been regarded as an important gas transmitter playing vital role in cytoprotective processes and redox signaling. It is very meaningful to monitor and analyze it in biosystem for obtaining important physiological and pathological information. Despite numerous fluorescent probes for cellular HS have been reported in past decades, only a few have capability to detect mitochondrial HS with near-infrared (NIR) emission. Therefore, a new mitochondria-targeting NIR fluorescent probe (Mito-NSH) for detection of cellular HS was developed by introducing 2,4-dinitrophenyl ether into a novel dye (Mito-NOH). A large "turn-on" NIR fluorescence response was obtained due to thiolysis of ether to hydroxyl group when Mito-NSH was treated with NaHS. Moreover, Mito-NSH could quantitatively detect HS at concentration ranging from 0 to 30 μM with a detection limit of 68.2 nM, and it exerts some superior optical properties, such as large stokes shift (107 nm), highly selectively mitochondria location, fast response and high selectivity to HS. More impressively, it was successfully applied to imaging exogenous and endogenously generated HS in living HeLa cells via confocal fluorescence microscopy.