Immobilization　of　enzymes　onto　metal-organic　frameworks　(MOFs)　through　a　biomimetic　mineralization　approach　can　preserve　biological　functionality　in　harsh　environments.　Despite　the　success　of　this　approach,　the　alkaline　environment　of　the　reaction　system,　which　is　caused　by　the　organic　monomers　of　MOFs,　makes　it　unsuitable　for　some　pH-sensitive　enzymes,　especially　for　trypsin.　Herein,　we　reported　a　facile　approach　for　the　one-pot　synthesis　of　trypsin-immobilized　magnetic　zeolite　imidazolate　framework-8　(iron　oxide@ZIF-8@trypsin),　where　the　growth　of　ZIF-8　around　the　citric　acid-modified　iron　oxide　and　immobilization　of　trypsin　occurred　simultaneously　when　the　pH　of　the　reaction　system　was　changed　to　some　extent.　With　a　large　specific　surface　area　and　a　high　enzyme　loading　capacity,　the　resultant　iron　oxide@ZIF-8@trypsin　exhibited　2.6　times　higher　enzymatic　activity　than　free　trypsin.　Moreover,　it　showed　a　favourable　magnetic　response　(43　emu　g−1)　which　made　the　operation　and　recycling　easy　and　convenient.　In　addition,　iron　oxide@ZIF-8@trypsin　could　be　applied　as　an　immobilized　enzyme　microreactor　(IMER)　to　rapidly　and　efficiently　digest　proteins　and　complex　human　serum　samples　with　satisfactory　results,　showing　great　promise　for　application　in　proteomic　analysis.　©　The　Royal　Society　of　Chemistry　2020.