We present a novel microfluidic magnetic bead separator for high-throughput applications. The system consists of an array of small Nd–Fe–B permanent magnets arranged into a checkerboard pattern with alternating magnetization directions and an array of integrated permalloy elements encapsulated in the bottom of a microfabricated separation chamber. The permanent magnets provide magnetic forces on millimeter-scale, whereas the integrated permalloy elements enhance local forces hundredfold on ∼10μm scale near the bottom of separation chamber. The fabrication of the chip is based on a multilayer SU-8 process combined with permalloy electroplating. The bead capturing behavior in a 10μL separation chamber was demonstrated using 250nm Nanomag-D^® plain silica beads. The capture efficiency of the system was quantified using vibration sample magnetometry. The experimental results show that magnetic beads can be captured with efficiencies of 91% and 54% at flow rates of 1mL/min and 4mL/min, corresponding to average linear fluid velocities of 1.7cm/s and 6.7cm/s, respectively. The integration of soft magnetic elements in the chip results in a slightly higher capturing efficiency. However, the distribution of captured beads is much more uniform in the system with soft magnetic elements than in that without soft magnetic elements. The results demonstrate that the magnetic bead separator is capable of high-throughput sample preparation, e.g., for cleaning inhibitors in DNA samples prior to PCR amplification.