Magnetic molecularly imprinted nanoparticles were prepared through surface-initiated reversible addition fragmentation chain transfer polymerization by using metronidazole as a template. The molecularly imprinted magnetic nanoparticles were characterized by attenuated total reflection Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, X-ray diffraction, and vibrating sample magnetometry. The adsorption characteristics were also investigated and the kinetics of the adsorption of metronidazole on the imprinted nanoparticles were described by the second-order kinetic model with the short equilibrium adsorption time (30min). The adsorption isotherm was well matched with the Langmuir isotherm in which the maximum adsorption capacity was calculated to be 40.1mg/g. Furthermore, the imprinted magnetic nanoparticles showed good selectivity as well as reusability even after six adsorption-desorption cycles. The imprinted magnetic nanoparticles were used as a sorbent for the selective separation of metronidazole from human serum. The recoveries of metronidazole from human serum changed between 97.5 and 99.8% and showed similar sensitivity as an enzyme-linked immunoassay method. Therefore, the molecularly imprinted magnetic nanoparticles might have potential application for the selective and reliable separation of metronidazole from biological fluids in clinical applications.