Novel 3-aminophenylboronic acid functionalized poly(glycidyl methacrylate-co-ethylene dimethacrylate) microspheres were prepared for the solid-phase extraction of glycopeptides/glycoproteins. The adsorption efficiency, maximum adsorption capacity, and specific recognition of the microspheres to glycoprotein were investigated. The results indicated excellent adsorption of glycoproteins by the microspheres, which are attributed to the well-defined boronic acid brushes on the microsphere surfaces. Furthermore, a solid-phase extraction microcolumn filled with the microspheres was used to efficiently enrich glycopeptides from enzymatic hydrolysates from human serum samples. The mass spectrometry results demonstrated that the method is suitable for the separation and enrichment of glycopeptides/glycoproteins from complex biological samples.