Unnatural amino acids are widely used in medicine,pesticide,material,and other industries and the green and efficient synthesis has attracted a lot of attention.In recent years,with the rapid development of synthetic biology,microbial cell factories have become a promising means for biosynthesis of unnatural amino acids.This study reviewed the construction and application of microbial cell factories for unnatural amino acid,including the synthetic pathway reconstruction,design/modification of key enzymes and their coordinated regulation with precursors,blocking of competitive alternative pathways,and construction of cofactor circulation systems.Meanwhile,on the basis of the new principles for designing the microbial cell factories,new biosynthetic pathways adapted to cells and the production environment,as well as new biomanufacturing system established based on cell adaptive evolution and intelligent fermentation regulation,we looked forward to the further construction and application of microbial cell factories for industrial bio-production.