AbstractProtein glycosylation is one of the most important PTMs in biological organism. Lectins such as concanavalin A (Con A) have been widely applied to N‐glycosylated protein investigation. In this study, we developed Con A‐immobilized magnetic nanoparticles for selective separation of glycoproteins. At first, a facile immobilization of Con A on aminophenylboronic acid‐functionalized magnetic nanoparticles was performed by forming boronic acid‐sugar‐Con A bond in sandwich structure using methyl α‐D‐mannopyranoside as an intermedium. The selective capture ability of Con A‐modified magnetic nanoparticles for glycoproteins was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line 7703. In total 184 glycosylated sites were detected within 172 different glycopeptides corresponding to 101 glycoproteins. Also, the regeneration of the protein‐immobilized nanoparticles can easily be performed taking advantage of the reversible binding mechanism between boronic acid and sugar chain. The experiment results demonstrated that Con A‐modified magnetic nanoparticles by the facile and low‐cost synthesis provided a convenient and efficient enrichment approach for glycoproteins, and are promising candidates for large‐scale glycoproteomic research in complicated biological samples.