This study demonstrated the feasibility of utilizing electrokinesis in an electrodeless dielectrophoresis chip to separate and concentrate microparticles such as biosamples. Numerical simulations and experimental observations were facilitated to investigate the phenomena of electrokinetics, i.e., electroosmosis, dielectrophoresis, and electrothermosis. Moreover, the proposed operating mode can be used to simultaneously convey microparticles through a microfluidic device by using electroosmotic flow, eliminating the need for an additional micropump. These results not only revealed that the directions of fluids could be controlled with a forward/backward electroosmotic flow but also categorized the optimum separating parameters for various microparticle sizes (0.5, 1.0 and 2.0 μm). Separation of microparticles can be achieved by tuning driving frequencies at a specific electric potential (90 Vpp·cm(-1)). Certainly, the device can be designed as a single automated device that carries out multiple functions such as transportation, separation, and detection for the realization of the envisioned Lab-on-a-Chip idea.