Artificial multienzyme biocatalysts have played a crucial role in biosynthesis because they allow for conducting complex reactions. Here, we reorted a facile approach to fabricate multienzyme nanodevices with high catalytic activity and stability based on protein assembly and chemical crosslinking. The self-assembled partner SpyCatcher and SpyTag were genetically fused with 2,3-butanediol hydrogenase and formate hydrogenase to produce Kg BDH-SC (SpyCatcher-fused 2,3-butanediol hydrogenase) and FDH-ST (SpyTag-fused formate hydrogenase), respectively. After assembling the two fusion proteins, the complexes were then immobilized on the functionalized silicon dioxide nanoparticles by glutaraldehyde, forming Kg BDH-SC-ST-FDH-SiO 2 with the capability of reducing 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol with cofactor regeneration. Under the optimal conditions, the created co-immobilized enzymes performed 49% activity recovery compared with the mixture of free enzymes as well as showed 2.9-fold higher catalytic activity than the traditional random co-immobilized enzymes. Moreover, Kg BDH-SC-ST-FDH-SiO 2 showed better pH stability and organic solvents stability than the free enzymes, and remained 52.5% overall catalytic activity after 8 cycles. Finally, the co-immobilized enzymes can reduce 60 mM HAP for fabrication of (R)-PED with cofactor regeneration in the phosphate buffer reaction system, affording 83.9% yield and above 99% optical purity. • Kg BDH-SC and FDH-ST can spontaneously link together by self-assembly. • Kg BDH-SC-ST-FDH-SiO 2 can asymmetrically prepare (R)-PED with cofactor regeneration. • Kg BDH-SC-ST-FDH-SiO 2 showed a higher catalytic activity than FDH- Kg BDH-SC-SiO 2. • Kg BDH-SC-ST-FDH-SiO 2 showed excellent pH stability and organic solvents stability. • Kg BDH-SC-ST-FDH-SiO 2 remained 52.5% of catalytic activity after 8 cycles. [ABSTRACT FROM AUTHOR]