Isolation of specific cells from whole blood is important to monitor disease prognosis and diagnosis. In this study, a vibration-assisted filtration (VF) device has been developed for isolation and recovery of specific cells such as leukocytes and pathogenic bacteria from human whole blood. The VF device is composed of three layers which was fabricated using injection molding with cyclic olefin copolymer (COC) pellets consisting of: a top layer with coin-type vibration motor (Ф = 10 mm), a middle plate with a 1 μm or 3 μm-pore filter membrane to separate of Staphylococcus aureus (S. aureus) cells or leukocytes (i.e. white blood cells) respectively, and a bottom chamber with conical-shaped microstructure. One milliliter of human whole blood was injected into a sample loading chamber using a 3 μm-pore filter equipped in the VF device and the coin-type vibration motor applied external vibration force by generating a rotational fluid which enhances the filtration velocity due to the prevention of the cell clogging on the filter membrane. The effluent blood such as erythrocytes, platelet, and plasma was collected at the bottom chamber while the leukocytes were sieved by the filter membrane. The vibration-assisted leukocyte separation was able to finish within 200 s while leukocyte separation took 1200 s without vibration. Moreover, we successfully separated S. aureus from human whole blood using a 1 μm-pore filter equipped VF device and it was further confirmed by genetic analysis. The proposed VF device provides an advanced cell separation platform in terms of simplicity, fast separation, and portability in the fields of point-of-care diagnostics.