Various industries ranging from water purification to pharmaceutical production have experienced multi separation steps that impose more process time and contamination possibility by batch operation. We propose a developed microfluidic particle sorter (miniaturized hydrocyclone) that adopts centrifugal force as it has ability to decline the number of separation steps and the risk of extrinsic contamination in continuous process. While biological industries have not relied on mini hydrocyclones considerably because of low efficiency and microfabrication difficulties, current work has been planned to conquer these obstacles. In this research, biomass separation from fermentation broth by 3 mm hydrocyclones was investigated. The effect of apex size, feed flow rate, hydrocyclone geometry were analyzed numerically in four mini-hydrocyclones. The most efficient mini-hydrocyclone was chosen to be made by elegant additive manufacturing technology and studied experimentally. The separation efficiency was achieved up to 90% while the concentration ratio of heavy stream (apex) to dilute stream (vortex finder) was reached more than twofold. The mini hydrocyclone performance in view of energy target was studied by Euler-Reynolds-Efficiency plots. The 4 μm cut size was achieved that is promising high throughput separation for biological particles.