In this study, for clearer inspection of biosamples, a Y-shaped microchannel with a downstream sudden expansion channel in which a series of asymmetric microelectrode pairs were embedded was designed to separate and trap the microparticles within NaCl (sodium chloride) liquid. NaCl liquid, with different sized particles, was injected through one inlet of the Y-shaped microchannel. In the pinched segment of the channel, the injected particles were pinched by another NaCl liquid introduced through another inlet of the Y-shaped microchannel and aligned along a sidewall. AC electric potentials were applied to each pair of asymmetric electrodes, generating a non-uniform electrical field resulting in small and large rolls on the electrodes. While the small particles passed through the electrodes, the particles were introduced into the rolls and trapped within the pairs of electrodes. Through the specific Y-shaped microchannel and the induced AC electroosmotic flow within the sudden expansion microchannel, separation and trapping of microparticles can be achieved continuously and simultaneously.