The development of methods to effectively capture N-glycopeptides from the complex biological samples is crucial to N-glycoproteome profiling. Herein, the hydrophilic chitosan-functionalized magnetic graphene nanocomposites (denoted as Fe3O4-GO@PDA-Chitosan) were designed and synthesized via a simple two-step modification (dopamine self-polymerization and Michael addition). The Fe3O4-GO@PDA-Chitosan nanocomposites exhibited good performances with low detection limit (0.4 fmol·μL-1), good selectivity (mixture of bovine serum albumin and horseradish peroxidase tryptic digests at a molar ration of 10:1), good repeatability (4 times), high binding capacity (75 mg·g-1). Moreover, Fe3O4-GO@PDA-Chitosan nanocomposites were further utilized to selectively enrich glycopeptides from human renal mesangial cell (HRMC, 200 μg) tryptic digest, and 393 N-linked glycopeptides, representing 195 different glycoproteins and 458 glycosylation sites were identified. This study provides a feasible strategy for the surface functionalized novel materials for isolation and enrichment of N-glycopeptides.