Hydrogen-bonded　organic　frameworks　(HOFs)　are　promising　carriers　for　enzyme　immobilization.　HOFs　suitable　for　enzyme　reactions　containing　nicotinamide　cofactors　and/or　larger　molecule　substrates　are　to　be　explored.　Herein,　we　report　the　first　example　of　nano-sized　mesoporous　HOFs　(nmHOFs)　for　in　situ　enzyme　immobilization.　Taking　tetrakis(4-amidiniumphenyl)　methane　(TAM)　and　1,3,6,8-tetrakis(p-benzoic　acid)　pyrene　(H4TBAPy)　as　building　blocks,　enzymes　induce　the　assembly　of　TAM　and　H4TBAPy　into　enzyme-nmHOF　(named　enzyme@TaTb)　in　aqueous　solution.　The　larger　π-conjugated　H4TBAPy　building　block　and　the　electrostatic/hydrogen-bonding　interactions　between　TAM　in　nmHOF　and　–COOH/–NH2　residues　in　enzymes　trigger　the　formation　of　TaTb　with　pore　aperture　of　2.4　nm　and　particle　size　from　19.9　±　6.4　to　56.4　±　20.1　nm.　As　a　demonstration,　lactate　dehydrogenase　(LDH)　that　can　convert　pyruvate　into　lactate　in　the　presence　of　NADH　is　immobilized　in　TaTb　nmHOF.　Compared　with　LDH@ZIF-8　and　LDH@Bio-HOF-1,　LDH@TaTb　affords　faster　diffusion　of　NADH　and　pyruvate,　thus　exhibiting　ultrahigh　activity　close　to　the　free　LDH.　Meanwhile,　the　exoskeleton　of　TaTb　nmHOF　is　capable　of　stabilizing　enzymes　through　spatial　confinement,　rendering　superior　stability　against　external　negative　stimuli.　The　TaTb　nmHOF　is　also　used　for　immobilizing　α-amylase　and　horseradish　peroxidase.　Our　study　may　facilitate　the　development　of　HOFs　into　platform　carriers　for　enzyme　immobilization.　©　2023　Elsevier　B.V.