A facile strategy based on the synergistic effect of molecular imprinting and boronate affinity was proposed for glycoprotein imprinting. Polydopamine (PDA)-coated boronic acid-functionalized molecularly imprinted silica nanoparticles (MIPs) were prepared by the “thiol–ene” click reaction using SiO₂ as the core, 3-acrylamidophenyl boronic acid (AAPBA) as the functional monomer, and horseradish peroxidase (HRP) as the glycoprotein template. A well defined core–shell structure of MIPs was obtained after self-polymerization of dopamine (DA) on the surface of HRP-immobilized silica nanoparticles (NPs). The polymerization conditions and adsorption behavior were investigated in detail in order to obtain the highest selectivity and binding capacity. Under the optimized conditions, the HRP-MIPs showed higher binding affinity towards HRP than non-imprinted nanoparticles (NIPs), and the corresponding adsorption capacity (Q) and imprinted factor (α) reached 0.58 μmol g⁻¹ and 2.6, respectively. The specificity for HRP recognition was evaluated with a competitive experiment, and the results indicated that the HRP-MIPs had higher selectivity for the template. The good features of the HRP-MIPs facilitated selective isolation and enrichment of trace HRP from human serum. In addition, the stability and regeneration were also investigated, which indicated that the HRP-MIPs had excellent reusability.