This work demonstrated the feasibility of a new particle trapping and separation approach, based on an EDEP-based microfluidic chip. Experimental observations and numerical simulations were facilitated to investigate the phenomena of EO or DEF depended on the driving frequencies at a given constant electric field. The results not only revealed the fluid cloud be generated a forward/backward EOF by adjusting the driving frequency, but also categorized the optimum trapping/separating frequencies for the different particle sizes. Besides, the proposed operating mode can be used to trap/separate particles and simultaneously convey them through the EDEP-based device, eliminating the need for an additional micropump. Certainly, the EDEP-based device can be envisioned as a single automated device that supports the multi-functions such as transportation, separation, and detection, facilitating the realization of a Lab-on-a-Chip.