Enzyme immobilization is essential to the commercial viability of various critical large‐scale biocatalytic processes. However, challenges remain for the immobilization systems, such as difficulties in loading large enzymes, enzyme leaching, and limitations for large‐scale fabrication. Herein, we describe a green and scalable strategy to prepare high‐performance biocatalysts through in situ assembly of enzymes with covalent organic frameworks (COFs) under ambient conditions (aqueous solution and room temperature). The obtained biocatalysts have exceptional reusability and stability and serve as efficient biocatalysts for important industrial reactions that cannot be efficiently catalyzed by free enzymes or traditional enzyme immobilization systems. Notably, this versatile enzyme immobilization platform is applicable to various COFs and enzymes. The reactions in an aqueous solution occurred within a short timeframe (ca. 10–30 min) and could be scaled up readily (ca. 2.3 g per reaction). In situ assembly of enzymes and covalent organic frameworks (COFs) enables the environmentally benign large‐scale fabrication of a new generation of high‐performance biocatalysts. This approach shows how limitations in enzyme immobilization can be overcome and it opens up a new avenue for the scalable fabrication of high‐performance biocatalysts to accelerate enzyme industrialization.