Selective　enrichment　of　peptides　from　complex　biosamples　is　essential　for　mass　spectrometry-based　proteomics　but　still　remains　a　challenge.　In　this　work,　a　facile　approach　was　developed　for　rapid　room-temperature　synthesis　of　core-shell　structured　magnetic　covalent　organic　framework　composite　nanospheres　(denoted　as　Fe3O4@TbBd)　by　using　monodisperse　Fe3O4　nanoparticles　as　the　magnetic　core　and　1,3,5-triformylbenzene　(Tb)　and　benzidine　(Bd)　as　two　building　blocks　in　the　presence　of　dimethyl　sulfoxide　(DMSO).　The　resultant　core-shell　structured　Fe3O4@TbBd　nanospheres　exhibited　high　adsorption　capacity　(28.5　mg　g-1),　fast　adsorption　kinetics　(2　g-1),　large　pore　volume　(0.63　cm3　g-1),　narrow　pore　size　distribution　(∼2.8　nm),　strong　magnetic　response　(41.5　emu　g-1),　as　well　as　good　thermal　and　chemical　stability.　Moreover,　the　Fe3O4@TbBd　nanospheres　also　showed　good　selectivity　towards　hydrophobic　peptides　and　a　size-exclusion　effect　against　proteins　due　to　the　inherent　π-π　stacking　interaction　and　interconnected　mesoporous　channels　of　covalent　organic　framework　(COF)　shells.　Taking　advantage　of　these　composite　nanospheres,　selective　extraction　and　efficient　enrichment　of　low　abundance　hydrophobic　peptides　from　human　serum　in　the　presence　of　high　abundance　proteins　were　achieved.　Based　on　the　HPLC-Q-TOF/MS　results,　29　hydrophobic　peptides　assigned　to　12　proteins　were　clearly　identified　in　5　ng　μL-1　human　serum　digestion　upon　treatment　with　Fe3O4@TbBd　nanospheres,　much　better　than　those　obtained　without　treatment,　confirming　the　outstanding　performance　of　the　Fe3O4@TbBd　nanospheres　in　proteome　analysis.　©　2017　The　Royal　Society　of　Chemistry.