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In this paper, we report a disposable on-chip blood serum separator using self-assembled silica microsphere filter, which can passively separate serum from human blood. A self-assembly packing method was developed for packing silica microspheres through a microfluidic channel on cyclic olefin copolymer (COC) substrate. Using the self-assembled microsphere filter, serum has been successfully separated from diluted human blood. The filters realized in this work are suitable for incorporating into a disposable polymer lab-on-a-chip, which can perform point-of-care testing (POTC) for clinical diagnostics using human blood.