Fast　and　highly　efficient　digestion　of　proteins　is　essential　for　high-throughput　proteomic　analysis.　Herein,　a　facile　approach　was　developed　for　self-assembly　preparation　of　trypsin-immobilized　capillary　monolithic　column　and　its　application　as　an　immobilized　enzyme　microreactor　(IMER)　for　fast　and　highly　efficient　proteolysis　was　described.　The　performance　of　the　trypsin-immobilized　monolithic　enzyme　microreactor　was　evaluated　by　in-situ　digestion　of　model　proteins.　The　results　showed　that　the　trypsin-immobilized　monolithic　enzyme　microreactor　had　much　higher　tryptic　digestion　efficiency　than　the　free　trypsin　in　solution,　where　the　coverage　of　peptide　sequences　by　mass　spectrometry　(MS)-based　analysis　could　bear　comparison　with　the　free　one,　while　the　digestion　time　was　dramatically　shortened　from　12　h　to　16　s.　Furthermore,　the　trypsin-immobilized　monolithic　enzyme　microreactor　also　exhibited　good　practicability　to　complex　human　serum　sample,　in　which　the　total　of　45　peptides　from　human　serum　albumin　(HSA)　matched　with　sequence　coverage　of　75%　were　precisely　identified.　The　successful　application　demonstrated　the　promising　potential　of　the　trypsin-immobilized　capillary　monolithic　column　as　the　IMER　in　high-throughput　proteomic　analysis.　©　2020