Biological particle separation has wide applications in medical diagnosis, bioengineering, and various other domains. Traditional methods, such as filtration, density gradient centrifugation, and size exclusion chromatography, face many challenges, including low separation resolution, low purity, and the inability to be seamlessly integrated into continuous processes. The development of microfluidics has paved the way for efficient and precise biological particle separation. Microfluidic chip‐based methods can generally be performed continuously and automatically, and microfluidic chips can integrate multilevel operations, including mixing, separation, detection, and so forth, thereby achieving continuous processing of particles at various levels. This review comprehensively investigates biological particle separation techniques based on microfluidic chips. According to the different sources of force effect on the particles during the separation process, they can be divided into active separation, passive separation, and affinity separation. We introduce the principles and device design of these methods respectively, and compare their advantages and disadvantages. For the introduction of each method, we used the most classic and latest research cases as much as possible. Additionally, we discussed the differences between experimental standard particles and biological particles. Finally, we summarized the current limitations and challenges of existing microfluidic separation techniques, while exploring future trends and prospects.