Separation of micro- and nano-sized biological particles, such as cells, proteins, and nucleotides, is at the heart of most biochemical sensing/analysis, including in vitro biosensing, diagnostics, drug development, proteomics, and genomics. However, most of the conventional particle separation techniques are based on membrane filtration techniques, whose efficiency is limited by membrane characteristics, such as pore size, porosity, surface charge density, or biocompatibility, which results in a reduction in the separation efficiency of bioparticles of various sizes and types. In addition, since other conventional separation methods, such as centrifugation, chromatography, and precipitation, are difficult to perform in a continuous manner, requiring multiple preparation steps with a relatively large minimum sample volume is necessary for stable bioprocessing. Recently, microfluidic engineering enables more efficient separation in a continuous flow with rapid processing of small volumes of rare biological samples, such as DNA, proteins, viruses, exosomes, and even cells. In this paper, we present a comprehensive review of the recent advances in microfluidic separation of micro-/nano-sized bioparticles by summarizing the physical principles behind the separation system and practical examples of biomedical applications.