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 en-zymes can be promoted, resulting in enhanced overall reaction efficiency. Inspired by nature, a variety of ap-proaches 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 mecha-nistic 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 mech-anistic 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 re-search progress has revealed the great biotechnological potential of artificial multi-enzyme complexes as a pow-erful tool for biomanufacturing.