Two-photon excitation fluorescence microscopy was combined with the two-phase microflow system in order to measure the fast interfacial reaction rate at liquid/liquid interfaces. The lactone cleavage kinetics of octadecylrhodamine B (C18RB) at the toluene/water and heptane/water interfaces was studied by this new method. The organic solution containing the nonfluorescent lactone of C18RB was made to flow as an inner flow with an aqueous outer sheath flow. The diameter of the inner flow was <20 mum. A focused fundamental beam of a Ti:sapphire pulse laser of 780 nm was irradiated to the interface, and emitted fluorescence from the fluorescent product was detected by a charge-coupled device (CCD) camera or a streakscope. The increase in the concentration of the fluorescent form of C18RB was measured along the interface of the inner flow of the toluene/water and heptane/water systems for 80 Its just after the contact of two phases. The analysis made by the time-dependent Langmuir adsorption model with the aid of the digital simulation method gave the cleavage reaction rate constants of the lactone form of C18RB at the liquid/liquid interfaces.