A　novel　imprinting　strategy　using　reversible　covalent　complexation　of　glycoprotein　was　described　for　creating　glycoprotein-specific　recognition　cavities　on　boronate-functionalized　monolithic　column.　Based　on　it,　a　molecularly　imprinted　monolithic　column　was　prepared　by　self-polymerization　of　dopamine　(DA)　on　the　surface　of　4-vinylphenylboronic　acid　(VPBA)-based　polymeric　skeletons　after　reversible　immobilization　of　horseradish　peroxidase　(HRP).　Due　to　the　combination　of　boronate　affinity　and　surface　imprinting　of　DA,　the　stable　and　accessible　recognition　sites　in　the　as-prepared　imprinted　monolith　could　be　obtained　after　the　removal　of　the　template,　which　facilitated　the　rebinding　of　the　template　and　provided　good　reproducibility　and　lifetime　of　use.　The　recognition　behaviors　of　proteins　on　the　bare　VPBA-based,　HRP-imprinted　and　nonimprinted　monolithic　columns　were　evaluated　in　detail　and　the　results　showed　that　the　HRP-imprinted　monolith　exhibited　higher　recognition　ability　toward　the　template　than　another　two　monolithic　columns.　Not　only　nonglycoproteins　but　also　glycoproteins　can　be　well　separated　with　the　HRP-imprinted　monolith.　In　addition,　the　feasibility　of　the　HRP-imprinted　monolith,　adopted　as　an　in-tube　solid　phase　microextraction　(in-tube　SPME),　was　further　assessed　by　selective　extraction　and　enrichment　of　HRP　from　human　serum.　The　good　results　demonstrated　its　potential　in　glycoproteome　analysis.　©　2013　Elsevier　B.V.