The　flexible　and　controlled　synthesis　of　metal-organic　framework　(MOF)-derived　hybrid　nanostructures　is　of　great　significance　in　fine　tuning　of　their　enrichment　performance　in　large-scale　and　in-depth　phosphoproteome　analysis.　Herein,　a　magnetic　guanidyl-functionalized　MOF　hybrid　coating　with　multiaffinity　sites,　denoted　as　Fe3O4@G-ZIF-8,　was　fast　fabricated　via　a　one-pot　epitaxial　growth　strategy　for　the　first　time　and　applied　for　selective　and　highly　efficient　enrichment　of　global　phosphopeptides.　The　intrinsic　unsaturated　metal　sites　of　ZIF-8　endow　the　surface-mounted　MOF　coatings　with　immobilized　metal　ion　affinity　chromatography　interaction　with　multiphosphorylated　peptides.　The　oriented　anchoring　of　bifunctional　guanidineacetic　acid　on　the　magnetic　MOF　nanospheres　provides　additional　affinity　sites　(guanidyl　groups)　for　specific　recognition　of　phosphopeptides　by　＇salt　bridge＇interaction,　as　well　as　active　site　carboxyl　groups　for　the　coordination　with　the　metal　ions.　The　as-prepared　Fe3O4@G-ZIF-8　exhibits　large　surface　area　(382.5　m2　g-1),　good　superparamagnetic　property　(41.6　emu　g-1)　and　stability,　and　size-exclusion　effect　(1.73　nm),　which　can　serve　as　a　specific　adsorbent　for　global　phosphopeptide　analysis　with　satisfactory　selectivity,　great　detection　sensitivity　(1　fmol),　and　rapid　magnetic　separation.　Moreover,　the　successful　application　of　Fe3O4@G-ZIF-8　for　selective　capture　of　both　multi-　A　nd　mono-phosphopeptides　from　human　saliva　and　serum　demonstrated　the　great　potential　of　magnetic　surface-mounted　MOF　coatings　in　effective　identification　of　low-abundance　phosphopeptides　by　matrix-assisted　laser　desorption　ionization　time-of-flight　mass　spectrometry　from　complicated　biological　matrices.　©　2022　American　Chemical　Society.