A real-time monitoring of the diffusion coefficient using a micro sensing device is valuable for analyzing the dynamic change of protein-protein interactions and the protein conformation, such as the molecular size and the higher order structure. In the present study, we have developed a novel micro-optical diffusion sensor (MODS) based on a laser-induced dielectrophoresis (LIDEP) enabling small sample volume and high-speed measurement. This paper reports the measurement principle, chip design, and the validity of the proposed method. MODS consists of a pair of transparent electrodes and a photoconductive layer sealing the liquid sample. AC voltage is applied between transparent electrodes, and two excitation lasers are intersected on the photoconductive layer. The electrical conductivity distribution of the a-Si:H layer due to the photoconductive effect generates a non-uniform electric field followed by the dielectrophoresis (DEP), and then the concentration distribution is induced by LIDEP force. After cutting the AC voltage, the mass diffusion is occurred, and the diffusion coefficient can be obtained by observing the one dimensional diffusion process along with the interference fringe pattern. In the preliminary measurement, the prototype of the DEP cell was fabricated by the micro electro mechanical systems (MEMS) technique in order to verify the applicability of MODS, and we confirmed the lattice-shaped concentration distribution of polystyrene beads in distilled water. The decay time of the diffusion of the concentration distribution agreed well with the theoretical calculation. As a result, the applicability of MODS as the diffusion coefficient measurement method was verified.