Highly　efficient　enrichment　of　N-glycopeptides　from　complicated　biosamples　based　on　mass　spectrometry　is　essential　for　biomedical　applications,　especially　in　disease　biomarker　research.　In　this　work,　glutathione　(GSH)-modified　hierarchical　flower-like　hollow　covalent　organic　frameworks　loaded　with　Au　nanoparticles　(HFH-COFs@Au@GSH)　were　synthesized　for　N-glycopeptide　enrichment.　Due　to　the　abundant　accessibility　sites,　high　specific　surface　area,　and　inherent　high　stability　of　the　hierarchical　flower-like　hollow　structure,　a　large　number　of　Au　NPs　and　hydrophilic　GSH　can　be　modified　on　the　HFH-COFs.　The　HFH-COFs@Au@GSH　displayed　excellent　hydrophilicity　and　remarkable　enrichment　performance　for　N-glycopeptides:　low　detection　limit　(0.1　fmol　μL−1),　large　adsorption　capacity　(200　μg　mg−1),　great　selectivity　(1　:　1000,　HRP　to　BSA),　and　good　reusability　(at　least　5　times).　Furthermore,　the　HFH-COFs@Au@GSH　were　successfully　applied　to　capture　N-linked　glycopeptides　in　human　serum,　and　308　N-glycosylation　peptides　corresponding　to　84　N-glycosylation　proteins　with　123　N-glycosylation　sites　were　detected.　Gene　ontology　analyses　were　used　to　elucidate　the　cellular　component,　biological　process　and　molecular　function　of　detected　glycoproteins　in　human　serum,　demonstrating　the　great　potential　of　the　HFH-COFs@Au@GSH　in　N-glycopeptide　enrichment　for　glycoproteomic　analysis　of　complex　biological　samples.　©　2022　The　Royal　Society　of　Chemistry.