<p>With the advancement of cell culture technology, the development of serum-free media (SFM) has become an important branch of modern biomedical research. This paper reviews the evolution from traditional fetal bovine serum (FBS)-dependent media to animal-derived (NAO)-free, protein-free (PFM), and chemically defined (CDM) media. These SFM not only address ethical and safety concerns in the use of FBS but also improve experimental reproducibility and reduce the risk of biological contamination. We highlight the applications of SFM in three major areas: cell therapy, gene therapy, and the production of vaccines and biological products. In these fields, SFM enables safer, scalable, and GMP-compliant manufacturing of cells, viral vectors, and recombinant proteins. Finally, we discuss future directions in medium development, emphasizing fully defined, customizable formulations to meet clinical and industrial needs. With this article, we provide a new perspective for researchers and developers to optimize the formulation of serum-free culture media to facilitate their widespread adoption in biomedical research and applications.</p> Graphical Abstract <p></p>