We report a new two-step microfluidic device that performs efficient, continuous, and high throughput small particle separation. The separation mechanism is based the on combined effects of inertial focusing in a spiral microchannel followed by particle deflection in a straight microchannel. The particle trajectory was studied using 1 and 5.5 μm fluorescent polystyrene microbeads as model particles to be separated. We demonstrate that at the condition of 0.4 ml/min inlet, 0.15 ml/min acting, and 0.02 ml/min protecting flow rates, the separation efficiencies of 1 μm particles and 5.5 μm particles were 99.7% and 98.3%, and the corresponding purities were 95.7% and 99.6%, respectively. This new design shows a significant separation performance for small particles, and therefore can be potentially integrated as a promising tool to separate rare of-interest particles, such as bacteria from homogenized food matrix as part of sample preparation procedure for food safety inspections.