DC-Dielectrophoresis (DC-DEP), the induced motion of the dielectric particles in a spatially non-uniform DC electric field, is applied to separate biological cells by size. The locally non-uniform electric field is generated by an insulating hurdle fabricated within a PDMS microchannel. The cells experience a negative DEP (accordingly a repulsive) force at the corners of the hurdle where the gradient of local electric-field strength is the strongest. The DC-DEP force acting on the cells is proportional to the cells’ size. Thus the moving cells deviate from the streamlines and the degree of deviation is dependent on the cell size. In this paper, we demonstrated by using this method that, combined with the electroosmotic flow, mixed biological cells of a few to tens of micrometers difference in diameter can be continuously separated into different collecting wells. For separating target cells of a specific size, all that is required is to adjust the voltage outputs of the electrodes.