This paper presents a microfluidic chip with arcshape electrodes array (ASEA) for separation of different-size particles/Cells based on negative dielectrophoresis (nDEP). The purpose of ASEA is to generate the dielectrophoretic force perpendicular to the microchannel direction within the gap between each pairs of arc-shape electrodes. The DEP chip consisted of two regions of ASEA; first ASEA was design for aligning all particles floating along with the wall of microchannel before enter the second ASEA region; the configuration of second ASEA was upside down with first region, thus, the DEP force could move particles from the one side of microchannel toward the other side with different magnitude corresponding to its size, consequently, different size of particles can be separated within the second ASEA region. In this study, three typically electrode shapes, trapezoidal shape, convex-arc shape and concave-arc shape, were investigated for constructing an electric field gradient along the perpendicular direction to fluid flow by numerical simulation. A convex arc-shape electrode can provide better separation effect due to the larger effective area of DEP force and relative small variation of VE2 as indicated in the simulation results. Therefore, we fabricated a PDMS microchannel with two convex ASEAs for demonstration of the separation function between 19pm and 8pm polystyrene particles. This method can provide a continuous and high throughput separation of different-size bioparticles once they have similar dielectric properties that cannot separated by traditional DEP approach.