This study presents a new method for classifying the sizes of colloidal nanoparticles of below 100 nm in diameter in liquid dispersion using a microchannel size exclusion chromatography (SEC) chip. This chip can classify polydisperse colloidal nanoparticles containing a mix of two monodisperse nanoparticles into several monodisperse particle populations. The particles classified by the SEC chip are then sequentially analyzed by a photon correlation spectroscopy (PCS) method in combination with a flow cell. Two different pillar patterns of such SEC chips were used in experiments to investigate the effects of these patterns on the nanoparticle classification performance. The results obtained were compared with those from a numerical simulation. Standard polystyrene latex particles with diameters of 20 nm and 100 nm were used in this study. The usefulness of this methodology was verified since the simulation and measurement results were in good agreement with each other.