In this paper, we present the design and experimental results of a multi-layered microfluidic electromagnetic cell separation device. Our channel consists of top and bottom layers in order to separate magnetically labeled cells in the vertical direction. Rapid separation of magnetic beads in top and bottom channel can be used in high throughput screening to monitor the efficacy and drug compounds. The experiments using the device were carried out with 4.5|j.m magnetic bead and magnetic labeled Jurkat cell under electromagnetic field of 1.55mT. Without the magnetic field, the magnetic labeled cells started to flow from the bottom inlet and exit out of the bottom channel outlet. In the presence of the magnetic field, the cells started in bottom channel are attracted upward by the electromagnetic field and flow through the top layered. Finally, the labeled cells flow out the top channel outlet. The separation efficiencies of the multi-layer structured microfluidic channel showed more than 95%. We found that the multi-layer structured microfluidic channel was very effective in enhancing the separation. This microfluidic channel can be potentially applied to Lab-on-a-chip system because of its attractive features such as high throughput, continuous sorting, simple and rapid fabrication.