A novel microfluidic device containing a bi-gap electrode pair is presented in this paper, and it is capable of continuously separating three different populations of particles using dielectrophoresis. A mixture of 5, 10, and 20 μm polystyrene particles is focused by a sheath flow and then separated based on size after flowing over a bi-gap electrode pair. A new solver is developed in OpenFOAM to investigate the effects of various parameters such as the flow rate, gaps, and electrode pair angles to achieve an appropriate configuration of the bi-gap electrode pair for efficient particle separation. Based on the numerical simulation results, three different configurations of bi-gap electrode pairs are fabricated. The paths of three populations of polystyrene particles under various operating conditions are experimentally examined and compared with numerical results. Then, by examining the purity of the separated particles with three different electrode configurations at different flow rates, the performance of the device is experimentally investigated. The results showed that by employing the proposed electrode configuration, at a maximum flow rate of 100 μL h-1 (25 μL h-1 for the sample), particles are separated precisely (with more than 99% purity for all particles at desired outlets) using a 20 Vpp sinusoidal electric potential with a frequency of 100 kHz. This novel microfluidic device is thus a practical device for continuously separating three different populations of particles/cells according to size in a heterogeneous admixture.