Antibody-free analysis is a potential method for glycoprotein analysis, but the development of this method has been limited by its unfavorable selectivity in recent years. Magnetic molecular imprinting, which integrates the fast separation of magnetic materials with high selectivity towards templates in molecular imprinting, was expected to be an effective sample pretreatment in antibody-free analysis for glycoproteins. However, the aggregation of magnetic imprinted nanoparticles and thick molecularly imprinted polymer (MIP) shells on the surface of magnetic carriers caused an unfavorable adsorption capacity, and unsatisfactory rebinding and elution rates, and has limited its application in glycoprotein extraction. Thus, highly dispersed magnetic molecularly imprinted nanoparticles (MMINs) with a well-defined thin film for the selective extraction of glycoprotein HRP were developed in this work. A solvothermal method was used in this work to improve the dispersity of Fe₃O₄ NPs (nanoparticles) and the MMINs. The thickness of the MIP film was optimized to provide the optimum extraction efficiency. Thus the adsorption capacity of the MMINs, the rebinding rate and the elution rate of the templates were greatly improved. As a result, the prepared MMINs not only exhibited excellent selectivity and high adsorption capacity to HRP, and an outstanding tolerance for interference, but also showed excellent rebinding and elution rates for extraction application. Furthermore, this method provided a reliable way to improve conventional magnetic molecular imprinting, and showed great potential for the analysis of glycoprotein tumor biomarkers in clinics in the future.