A turn-on spiropyran functionalized fluorescein derivative (FMC) is developed for targeting HS- in mitochondria. FMC exhibits very weak fluorescence at 525 nm under the excitation of 470 nm in aqueous solution due to its colorless spiropyran form; upon addition of HS-, a strong fluorescence enhancement by 6.4-fold is observed with spirocycle-opened merocyanine form and rapid trapping kinetics for HS-. FMC has good biocompatibility and high selectivity towards HS- with a detection limit of 88.2 nM and is very sensitive among the reported H2S fluorescent probes. Moreover, the significant colocalization of FMC with Mito Tracker® Deep Red FM in human laryngeal epidermoid carcinoma (HEp-2) cells and the Pearson correlation coefficient of 0.87 together demonstrate that FMC can target and image the endogenous H2S in the mitochondria of living cells.