Multi-enzyme complexes are the results of natural evolution to facilitate cascade biocatalysis. Through enzyme colocalization within a complex, the transfer efficiency of reaction intermediates between adjacent cascade enzymes can be promoted, resulting in enhanced overall reaction efficiency. Inspired by nature, a variety of approaches have been developed for the assembly of artificial multi-enzyme complexes with different spatial organizations, aiming at improving the catalytic efficiency of enzyme cascade. A recent trend of this research area is the creation of enzyme complexes with a controllable spatial organization which helps with the mechanistic studies and bears the potential to further increase metabolic productivity. In this review, we summarize versatile strategies for the assembly of artificial multi-enzyme complexes, followed by an inspection of the mechanistic studies of artificial multi-enzyme complexes for their enhancement of catalytic efficiency. Furthermore, we provide some highlighted in vivo, ex vivo, and in vitro examples that demonstrate the ability of artificial multi-enzyme complexes for enhancing the overall production efficiency of value-added compounds. Recent research progress has revealed the great biotechnological potential of artificial multi-enzyme complexes as a powerful tool for biomanufacturing. (C) 2020 The Chemical Industry and Engineering Society of China, and Chemical Industry Press Co., Ltd. All rights reserved.