We report herein a two-photon ratiometric fluorescent probe (DNEPI) for mitochondrial cysteine (Cys) detection on the basis of a merocyanine (compound 1) as the two-photon fluorophore and a 2,4-dinitrobenzensulfonyl (DNBS) unit as the biothiol reaction site. Upon reaction with Cys in DMSO/PBS (1/1, v/v), DNEPI showed a distinct ratiometric fluorescence emission characteristic (F583 nm/F485 nm) linearly proportional to Cys concentrations over the range of 2-10 μM, which was attribute to the enhanced intramolecular charge transfer (ICT) effect by cleavage of the sulfonic acid ester bond of DNEPI to release compound 1. More importantly, the probe could detect Cys with a fast response time (within 2 min) and the detection limit was quantitatively calculated as 0.29 μM. Furthermore, DNEPI not only exhibited high selectivity toward Cys over other similar biothiols, including homocysteine (Hcy) and glutathione (GSH), but also displayed significant mitochondrial-targeting ability, which were favorable for mitochondrial Cys-selective imaging. Subsequently, application of DNEPI to Cys imaging in live cells was successfully achieved by two-photon fluorescence microscopy, suggesting that the probe proposed here could be used to monitor mitochondrial Cys concentration changes in live cells with negligible interference from other biological thiols.