In this paper, a PMMA (polymethylmethacrylate) microfluidic device with filtration features fabricated by hot embossing and thermal bonding was used to separate RBCs (red blood cells) from whole rat blood. The filtration features are composed of 20 µm deep and 300 µm wide main channels, 15 µm high and 25 µm wide micro-dams which were fabricated in main channels and an array of orthogonal side channels for perfusion flow to collect RBCs. As rat blood advances through the main channels, a perfusion flow through the side channels washes away RBCs which are sufficiently small to enter the gaps between the micro-dams and the cover plate. A silicon mold fabricated by dry etching was used to produce three-dimensional filtration features on PMMA substrates. Oxygen plasma treatment was used to increase the adhesive ability of PMMA surfaces, which enables thermal bonding at 86 °C and 0.75 MPa. The distortion of microchannels and micro-dams has been minimized, which makes the value of the gap between the micro-dam and the cover plate appropriate for cell filtration.