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The present invention relates to a microfluidic separation apparatus, a separation method using the same, and a separation kit for circulating rare cells in blood comprising the same. More particularly, the present invention relates to a microfluid-based separation technique which fixates targeted particles having a specific affinity to magnetic nanoparticles treated in a mixture sample to the inside of the apparatus through a magnetic body, and obtains a sample with targeted particles removed. The microfluidic separation apparatus can be effectively used for removing white cells from a blood sample to obtain circulating rare cells (CRC) and, particularly, circulating tumor cells (CTC). The microfluidic separation apparatus of the present invention comprises : a mixed channel (100); an incubation channel (200); and a separation channel (300). The microfluidic separation apparatus of the present invention can minimize the loss of particles subject to extraction and can easily perform separation with high efficiency and high speed by continuously and automatically performing a series of pre-treatment processes such as separation of a sample, etc., in a single microfluidic chip.(AA) Magnetic nanoparticle(BB) SpecimenCOPYRIGHT KIPO 2016