Plants generate flavonoids, which are minuscule molecular secondary metabolites that have various biological roles, such as anti-carcinogenic, anti-inflammatory, and antioxidant activity. It may be found in medicinal herbs, fruits, and drinks, including tea, wine, and vegetables. Biotechnology provides several in vitro techniques for utilizing such active substances, notably callus cultures and cell suspension cultures, that have recently emerged in biotechnology sectors for investigating and improving the generation of these substances. Transformation processes, including hairy root culture, are also frequently employed for flavonoid synthesis. Nano-treatment is a revolutionary technique to increase flavonoid synthesis. The current review focuses on the biochemistry of flavonoids, biosynthesis regulations, and the different strategies for in vitro flavonoid production.