Immobilization　of　fragile　enzymes　is　vital　to　expanding　its　application　in　the　extracellular　environment.　Covalent　organic　frameworks　(COFs),　as　a　class　of　emerging　porous　materials,　are　promising　platforms　for　enzyme　immobilization　owing　to　their　high　porosity　and　tunable　structure.　However,　the　interior　pores　of　COFs　often　fail　to　play　their　roles　because　of　inaccessibility,　resulting　in　decreased　performance　of　immobilized　enzymes.　Here,　we　introduce　a　facile　approach　to　construct　hierarchically　macro-mesoporous　COF　(Hm-COF)　via　a　template-assistant　strategy.　The　mesoporous　channels　of　Hm-COF　were　used　to　entrap　cytochrome　c　(Cyt　c),　and　the　macropores　of　Hm-COF　were　adopted　as　diffusion　channels　to　enhance　the　mass　transfer　of　guest　molecules.　The　resulting　Hm-COF　demonstrated　an　enhanced　uptake　capacity　of　Cyt　c　compared　with　mesoporous　COF　(m-COF).　Furthermore,　the　catalytic　activity　of　Cyt　c　immobilized　in　Hm-COF　(Cyt　c@Hm-COF)　was　remarkably　higher　than　that　of　Cyt　c　immobilized　in　m-COF　(Cyt　c@m-COF).　Taking　advantage　of　the　excellent　activity,　Cyt　c@Hm-COF　was　employed　as　the　sensor　for　the　sensitive　detection　of　dopamine　(DA).　This　study　opens　a　new　avenue　for　the　construction　of　COFs　with　hierarchical　pores　and　high-performance　immobilized　enzymes.　©　2025　Elsevier　B.V.