In the epitope imprinting method, a short peptide, even without a defined tertiary structure, can be used as the template to produce protein-selective polymers. In this study, His-tag-epitope imprinted magnetic nanoparticles (EMIP-MNPs) were prepared via epitope imprinting method onto the surface of the magnetic nanoparticles for specific recognition and separation of thyroid peroxidase antigens (TPO). 3-(Trimethoxysilyl) propyl methacrylate (TMSPM), copper (II) acrylate (Cu(II)Ac) and N-isopropylacrylamide (NIPAAm) were chosen as functional monomers. Ethyleneglycoldimethacrylate (EGDMA) and 2,2'-azobisisobutyronitrile (AIBN) were selected as crosslinking agent and initiator, respectively. The EMIP-MNPs were prepared within 30 min by epitope imprinting method which was shorter than the surface imprinting method. The prepared EMIP-MNPs were thermosensitive and showed selective affinity to target TPO antigens. Moreover, the EMIP-MNPs were easily and quickly recognized and separated to target antigens from the whole blood sample.