Highly　efficient　extraction　of　glycopeptides　prior　to　mass　spectrometry　detection　is　extremely　crucial　for　glycoproteomic　research,　especially　in　disease　biomarker　research.　Reported　here　is　the　first　time　by　applying　two-dimensional　(2D)　covalent　organic　framework　(COFs)　nanosheets　for　highly　efficient　enrichment　of　glycopeptides.　Particularly,　by　incorporating　hydrophilic　monomers　through　a　bottom-up　strategy,　the　2D　COF　nanosheets　(denoted　as　NUS-9)　displayed　an　ultra-high　graft　density　of　sulfonic　groups　and　super-hydrophilicity.　In　addition,　because　of　the　large　surface　area,　low　steric　hindrance,　high　chemical　stability,　and　abundant　accessibility　sites　of　2D　COF　nanosheets,　NUS-9　exhibited　remarkable　efficiency　for　glycopeptide　enrichment,　involving　excellent　detection　sensitivity　(0.01　fmol　μL−1),　outstanding　enrichment　capability,　and　good　enrichment　selectivity　(1:1500,　horseradish　peroxidase　(HRP)　tryptic　digest　to　bovine　serum　albumin　(BSA)　tryptic　digest),　and　recovery　(92.2　±　2.0%).　Moreover,　the　NUS-9　was　able　to　unambiguously　detect　631　endogenous　glycopeptides　from　human　saliva,　demonstrating　an　unparalleled　high　efficiency　in　glycopeptide　enrichment.　Gene　ontology　analyses　of　proteins　from　human　saliva　enriched　by　NUS-9　demonstrated　its　potential　for　comprehensive　glycoproteome　analysis.　©　2023　Elsevier　B.V.