White blood cells (WBCs) constitute only about 0.1% of human blood cells, yet contain rich information about the immune status of the body; thus, separation of WBCs from the whole blood is an indispensable and critical sample preparation step in many scientific, clinical, and diagnostic applications. In this paper, we developed a continuous and high-throughput microfluidic WBC separation platform utilizing the differential inertial focusing of particles in serpentine microchannels. First, separation performance of the proposed method is characterized and evaluated using polystyrene beads in the serpentine channel. The purity of 10-μm polystyrene beads is increased from 0.1% to 80.3% after two cascaded processes, with an average enrichment ratio of 28 times. Next, we investigated focusing and separation properties of Jurkat cells spiked in the blood to mimic the presence of WBCs in whole blood. Finally, separation of WBCs from human whole blood was conducted and separation purity of WBCs was measured by the flow cytometry. The results show that the purity of WBCs can be increased to 48% after two consecutive processes, with an average enrichment ratio of ten times. Meanwhile, a parallelized inertial microfluidic device was designed to provide a high processing flow rate of 288 ml/h for the diluted (×1/20) whole blood. The proposed microfluidic device can potentially work as an upstream component for blood sample preparation and analysis in the integrated microfluidic systems.