Plant flavonoids, a class of plant secondary metabolites with diverse physiological activities, hold great application potential in the food, pharmaceutical, cosmetics, and chemical industries. In recent years, the remarkable progress in omics technologies and synthetic biology has significantly advanced the elucidation of the natural biosynthetic pathways of flavonoids and the in-depth functional characterization of their key enzymes. This achievement serves as a crucial theoretical foundation for the construction of microbial cell factories. This review comprehensively outlines the mining strategies for identifying key enzymes in flavonoid biosynthetic pathways, elaborates on the functions of core enzymes and the progress in their engineering modification, and summarizes the latest progress in the microbial synthesis of flavonoid compounds. Moreover, this review analyzes the core bottlenecks in the large-scale production of flavonoids in microbial cell factories from the aspects of heterologous enzyme activity adaptation, metabolic network regulation, and product stability. Finally, it proposes the development direction of interdisciplinary integration and intelligent design, thereby providing theoretical support for the industrialization of flavonoid green biomanufacturing.