There has been an increase of research into μTAS. In general, detection with this device is based on a reaction between reactants in the fluid and on the wall of a microchannel. Therefore, it is important to elucidate physical phenomena in the near-wall region of the microchannel to achieve design optimization of the device. The aim of this study is experimental elucidation of single-nanoparticle behavior in microfiuid by observation with an evanescent illumination which allows three-dimensional location measurement of nanoparticles in the near-wall region. Instead of biological molecules, fluorescent nanoparticles (20 nm diameter) were employed for the single particle imaging. Since there are issues of a measurement accuracy due to considerably small diameter of nanoparticles for the imaging and low brightness of a single nanoparticle in the deeper location, an optimal setting of shooting and a novel algorithm of image processing were proposed. The result of experiments showed that perpendicular distribution of nanoparticle concentration exists.