<p>Vitamin B2 (riboflavin), a precursor of the essential coenzymes flavin mononucleotide and flavin adenine dinucleotide, plays a critical role in numerous cellular metabolic pathways, including redox reactions, energy production, and biosynthetic processes. Due to its fundamental importance in human and animal nutrition, riboflavin has found wide application across the food, pharmaceutical, nutraceutical, and cosmetic industries. In recent decades, the development of metabolic engineering techniques has revolutionized riboflavin production, transitioning from traditional chemical synthesis to environmentally sustainable microbial fermentation. Microorganisms such as <i>A. gossypii</i>, <i>Candida famata</i>, and <i>Bacillus subtilis</i> have been extensively engineered to enhance riboflavin yield at an industrial scale. Key metabolic engineering strategies include reprogramming the purine metabolic network, mutating ribulose-5-phosphate 3-epimerase, and relieving regulatory constraints on purine biosynthesis. These advances have significantly improved the efficiency, cost-effectiveness, and sustainability of industrial riboflavin production. This review article presents an in-depth analysis of biotechnological methods aimed at increasing riboflavin production, ensuring a consistent supply of this crucial micronutrient for both human and animal nutrition. It stands as a significant reference for researchers and industry stakeholders.</p>