Cell-free multi-enzyme cascade is a biomanufacturing platform for in vitro synthetic biology, located between microbial fermentation and enzyme molecular catalysis. Multi-enzyme molecular machines are unique biocatalysts for in vitro biotransformation. Based on the principle of simplifying complexity, multiple natural enzymes, artificial enzymes and (biomimetic) coenzymes are used to reconstruct in vitro artificial metabolic pathways, getting rid of the limitations of self-reproduction of organisms and transcending Cell synthesis limits, achieving important biochemical synthesis, efficient and low-cost production of bulk biochemical products, and efficiently reshaping the production potential of the "cell factory".