We investigate diffusion and mixing in a microchannel by using luminol chemiluminescence (CL) to estimate the local chemical reaction rate. The degree of mixing in micromixers is generally evaluated from the deviation of concentration profiles measured by fluorescence from a uniform concentration profile. The degree of mixing measured by this method is a macroscopic estimate, which is inappropriate for investigating diffusion and mixing in microchannels. In this study, the luminol CL reaction is used to visualize and quantitatively measure local diffusion and mixing at an interface between two liquids in a microchannel. Blue CL is observed where luminol reacts with hydrogen peroxide at the mixing layer. Diffusion and mixing in a microchannel are investigated by sequentially measuring the CL and fluorescence. The experimental results are compared with the results of a numerical simulation that involves solving transport equations including the chemical reaction term. By calibrating the CL intensity with the chemical reaction rate estimated by the numerical simulation, the local chemical reaction can be quantitatively estimated from the CL intensity profile.