Plants, fungi, and microorganisms are equipped with biosynthesis machinery for producing thousands of secondary metabolites. These compounds have important functions in nature as a defence against predators or competitors as well as other ecological significances. The full utilization of these compounds for food, medicine, and other purposes requires a thorough understanding of their structures and the distinct biochemical pathways of their production in cellular systems. In this review, flavonoids as classical examples of secondary metabolites are employed to highlight recent advances in understanding how valuable compounds can be regulated at various levels. With extensive diversity in their chemistry and pharmacology, understanding the metabolic engineering of flavonoids now allows us to fine-tune the eliciting of their production, accumulation, and extraction from living systems. More specifically, recent advances in the shikimic acid and acetate biosynthetic pathways of flavonoids production from metabolic engineering point of view, from genes expression to multiple principles of regulation, are addressed. Specific examples of plants and microorganisms as the sources of flavonoids-based compounds with particular emphasis on therapeutic applications are also discussed.