A deeper understanding of disease heterogeneity highlights the urgent need for precision medicine. Microfluidics, with its unique advantages, such as high adjustability, diverse material selection, low cost, high processing efficiency, and minimal sample requirements, presents an ideal platform for precision medicine applications. As nanoparticles, both of biological origin and for therapeutic purposes, become increasingly important in precision medicine, microfluidic nanoparticle separation proves particularly advantageous for handling valuable samples in personalized medicine. This technology not only enhances detection, diagnosis, monitoring, and treatment accuracy, but also reduces invasiveness in medical procedures. This review summarizes the fundamentals of microfluidic nanoparticle separation techniques for precision medicine, starting with an examination of nanoparticle properties essential for separation and the core principles that guide various microfluidic methods. It then explores passive, active, and hybrid separation techniques, detailing their principles, structures, and applications. Furthermore, the review highlights their contributions to advancements in liquid biopsy and nanomedicine. Finally, it addresses existing challenges and envisions future development spurred by emerging technologies such as advanced materials science, 3D printing, and artificial intelligence. These interdisciplinary collaborations are anticipated to propel the platformization of microfluidic separation techniques, significantly expanding their potential in precision medicine.