Continuous dielectrophoretic separation is recognized as a powerful technique for a large number of applications including early stage cancer diagnosis, water quality analysis, and stem-cell-based therapy. Generally, the prefocusing of a particle mixture into a stream is an essential process to ensure all particles are subjected to the same electric field geometry in the separation region. However, accomplishing this focusing process either requires hydrodynamic squeezing, which requires an encumbering peripheral system and a complicated operation to drive and control the fluid motion, or depends on dielectrophoretic forces, which are highly sensitive to the dielectric characterization of particles. An alternative focusing technique, induced charge electro-osmosis (ICEO), has been demonstrated to be effective in focusing an incoming mixture into a particle stream as well as nonselective regarding the particles of interest. Encouraged by these aspects, we propose a hybrid method for microparticle separation based on a delicate combination of ICEO focusing and dielectrophoretic deflection. This method involves two steps: focusing the mixture into a thin particle stream via ICEO vortex flow and separating the particles of differing dielectic properties through dielectrophoresis. To demonstrate the feasibility of the method proposed, we designed and fabricated a microfluidic chip and separated a mixture consisting of yeast cells and silica particles with an efficiency exceeding 96%. This method has good potential for flexible integration into other microfluidic chips in the future.