A fluorescent probe that enables ratiometric imaging of endogenous hypochlorous acid (HOCl) in cells and tissues by two-photon microscopy is developed based on a red-emitting acetyl-benzocoumarin (AcBC) dye. An oxathiolane group in the probe reacts with HOCl to generate the AcBC dye, which involves a ratiometric fluorescence change only toward HOCl along with high sensitivity.