Covalent　organic　framework　(COF)-based　monolithic　columns　have　been　extensively　used　for　sample　preparation.　In　order　to　maximize　the　COF　extraction　properties,　it　is　attractive　to　increase　the　COF　surface　coverage　of　the　COF-based　monolithic　column.　Herein,　a　novel　monolithic　column　with　multiple　COF　surface　layers　was　prepared　via　an　in-situ　surface　self-assembly　strategy　to　increase　the　COF　surface　coverage.　For　this　preparation,　the　poly　(p-phenylenediamine)　(PPDA)　fibers　were　first　grafted　onto　the　parent　monolith.　Then,　due　to　the　abundant　NH2-anchor　sites　of　the　PPDA　fiber,　a　large　number　of　COF　microspheres　were　self-assembled　on　PPDA　fibers,　and　further　interlinked　with　each　other　to　form　multiple　COF　surface　layers　on　the　monolith,　which　further　improve　the　COF　surface　coverage.　Various　characterization　techniques　were　employed　to　confirm　this　successful　preparation.　Benefiting　from　the　enhanced　COF　surface　coverage,　the　developed　monolith　exhibited　superior　extraction　ability　towards　benzodiazepines　(BZDs),　which　are　central　nervous　system　depressants　that　are　tightly　controlled　by　governments.　The　hydrophobic　and　π-π　stacking　interactions　should　be　the　main　adsorption　mechanism　for　in-tube　SPME　of　BZDs.　Under　the　optimal　SPME　conditions,　a　simple　in-tube　SPME-HPLC-DAD　method　was　established　for　the　detection　of　trace　BZDs　in　saliva,　with　a　wide　linear　range　(5　∼　300　ng/mL),　excellent　sensitivity　(LODs　≤　2　ng/mL)　and　satisfactory　recoveries　(81.4%　∼　98.0%).　This　research　provided　a　versatile　route　to　fabricate　the　COF-based　monolith　with　high　surface　coverage,　demonstrating　its　great　potential　in　efficient　sample　preparation.　©　2023　Elsevier　B.V.