This paper presents a microfluidic system for separation of microparticles based on the use of dielectrophoretic barriers, which are constructed by aligning two layers of microelectrode structure face-to-face on the top and bottom sides of the microchannel. The energized barriers tend to prevent the particles in the flow from passing through. However, particles may penetrate the barriers if a sufficiently high flow rate is used. The flow velocity at which the particles begin to penetrate the barrier is defined as threshold velocity. Different particles are of different threshold velocities so that they can be separated. In this paper, the electrodes are configured with open ends and aligned with a certain angle to the direction of the flow. Polystyrene microbeads of different sizes (i.e., 9.6 and 16 μm in diameter) are studied in the tests. Under the experimental conditions, two particle trajectories are observed: the 9.6 μm beads penetrate the barriers and move straightly toward the fluidic outlet, while the 16 μm beads snake their way along the electrode edges at a relatively low speed. The two subpopulations of particles are separated into spatial distance of ∼10 mm within tens of seconds. The system presents a rapid and dynamic separation process within a continuous flow.