The separation of particles from a complex mixture is important to a wide range of applications in industry, biology, medicine etc. This work demonstrates a microfluidic approach to separate similar-sized fluorescent and nonfluorescent particles based upon the difference in their surface charges inside a reservoir. Such a separation exploits the reservoir-based dielectrophoresis, which is induced by the inherent electric field gradient formed at the reservoir–microchannel junction, to isolate the trapped fluorescent particles within the reservoir from the streaming nonfluorescent particles. The effects of the DC field magnitude (or equivalently the electrokinetic flow magnitude) and the AC field frequency of DC-biased AC electric fields are investigated on particle separation. A numerical model is also developed to simulate the electrokinetic transport behaviors of the two types of particles. This demonstrated reservoir-based dielectrophoresis particle sorter can operate in parallel to increase the flow throughput. It is suitable for integration with other functional parts into lab-on-a-chip devices for diverse particle handling.