Highly　specific　capture　of　phosphopeptides,　especially　multi-phosphopeptides,　from　complex　biological　samples　is　critical　for　comprehensive　phosphoproteomic　analysis,　but　it　still　poses　great　challenges　due　to　the　lack　of　affinity　material　with　ideal　enrichment　efficiency.　Here,　two-dimensional　(2D)　covalent　organic　framework　(COFs)　nanosheets　was　applied　for　selective　separation　of　phosphopeptides　for　the　first　time.　Particularly,　by　incorporating　guanidinium　units,　the　2D　guanidinium-based　COF　nanosheets　(denoted　as　TpTGCl　CONs)　exhibited　controllable　and　specific　enrichment　performance　towards　global/multi-phosphopeptides.　TpTGCl　CONs　was　easy　to　prepare　and　showed　large　surface　area,　low　steric　hindrance,　abundant　accessible　interaction　sites　and　high　chemical　stability.　Taking　these　merits　together,　TpTGCl　CONs　exhibited　excellent　efficiency　for　phosphopeptide　enrichment,　such　as　low　detection　limits　(0.05　fmol　μL−1　for　global　phosphopeptides　and　0.1　fmol　μL−1　for　multi-phosphopeptides),　high　selectivity　(1:5000　of　molar　ratios　of　β-casein/BSA　for　both　global　and　multi-phosphopeptides),　high　adsorption　capacity　(100　mg　g−1　for　global　phosphopeptides　and　50　mg　g−1　for　multi-phosphopeptides).　Furthermore,　TpTGCl　CONs　could　be　reused　due　to　the　high　chemical　stability.　In　addition,　TpTGCl　CONs　were　successfully　applied　to　controllable　and　specific　capture　of　endogenous　global/multi-phosphopeptides　from　human　serum　and　human　saliva,　indicating　its　good　potential　in　rapid　and　sensitive　detection　of　biomarkers　from　biological　fluid.　Finally,　rat　liver　protein　digest　was　used　to　confirm　the　high　specificity　of　TpTGCl　CONs　towards　multi-phosphopeptides　and　demonstrated　its　potential　as　an　ideal　enrichment　probe　for　comprehensive　phosphoproteomic　analysis.　©　2021　Elsevier　B.V.