The ratiometric fluorescent probe B6S, which contains pyrene as a fluorophore and imidazoline-2-thione as a reactive site, was developed for detection of hypochlorite (OCl-). B6S displays a high specificity toward OCl- in contrast to other reactive oxygen species and reactive nitrogen species. The probe has a low detection limit and operates under biological conditions. Moreover, the low cytotoxicity of B6S enables it to be utilized effectively for OCl- imaging in living cells and tissues by using two-photon microscopy. The findings indicate that B6S has the capability of serving as a probe to explore the biological functions of OCl- in living systems.