Hypochlorite (OCl-) plays a key role in the immune system and is involved in various diseases. Accordingly, direct detection of endogenous OCl- at the subcellular level is important for understanding inflammation and cellular apoptosis. In the current study, a two-photon fluorescent off/on probe (PNIS) bearing imidazoline-2-thione as an OCl- recognition unit and triphenylphosphine (TPP) as a mitochondrial-targeting group was synthesized and examined for its ability to image mitochondrial OCl- in situ. This probe, based on the specific reaction between imidazoline-2-thione and OCl-, displayed a selective fluorescent off/on response to OCl- with the various reactive oxygen species in a physiological medium. PNIS was successfully applied to image of endogenously produced mitochondrial OCl- in live RAW 264.7 cells via two-photon microscopy.