We have found that photo-excited indigo carmine causes stepwise proton transfer to generate mono-alcohol intermediate, which immediately returns to the parent molecule [I. Iwakura, A. Yabushita, T. Kobayashi, Chem. Lett. 38 (2009) 1020]. The kinetic isotope effect in the proton transfer of this system has been studied by direct observation of the real-time molecular vibrational amplitude during the reaction including the transition state using a sub-5-fs laser pulse. The kaH / kaD ratio for the proton transfer after photo-excitation is observed to be 1.5 ± 0.1.