Widely used in cell culture and microbiological media to support growth, especially in organisms requiring biotin as a growth factor. Essential component in the production of therapeutic proteins and antibodies in biopharmaceutical manufacturing. Employed as a biochemical reagent in enzyme-linked immunosorbent assays (ELISA) and other immunoassays due to its strong binding affinity to streptavidin and avidin. Utilized in molecular biology for nucleic acid labeling, purification, and detection techniques such as blotting and sequencing. Common additive in vitamin supplements and nutritional formulations for its role in metabolic health. Used in cosmetics and personal care products to promote healthy skin, hair, and nails by supporting keratin production, the primary protein in hair and nails. Also applied in animal feed to improve metabolic function and overall health in livestock.

In medicine, D-Biotin is used to treat biotin deficiency, which may occur in individuals with malabsorption disorders or those on prolonged parenteral nutrition. It is also under study for high-dose applications in multiple sclerosis to enhance nerve cell function.