Development of high performance fluorescent chemosensors for the detection of ClO⁻ in vitro and in vivo is very desirable, because many human diseases are caused by ClO⁻. In this paper, a highly selectivity and sensitive fluorescent probe, EDPC, based on 3-acetylcoumarin, was synthesized, which could respond to ClO⁻ and exhibit an “off–on” mode in Tris–HCl buffer (pH = 7.2, 10 mM, 50% C₂H₅OH) solutions. The detection limit of the EDPC probe for ClO⁻ was as low as 1.2 × 10⁻⁸ M. Moreover, the high selectivity and high sensitivity of EDPC towards ClO⁻ are attributed to the oxidation reaction between the C–O of the coumarin lactone and the CC formed by aldol condensation and the mechanism was further verified using ESI-MS and DFT. Additionally, the concentrations of ClO⁻ in real water were also calculated using the EDPC probe and showed good recovery. Finally, the distribution of intracellular endogenous ClO⁻ was gained by confocal fluorescence microscopy in living HEK293T cells.