<p>Medicinal plants produce essential secondary metabolites which have immense potential in human welfare, including pharmaceutical and nutraceutical applications. Over the last few decades, hairy root cultures have emerged as a promising biotechnological platform for the sustainable synthesis of pharmacologically important secondary metabolites. This approach exploits the inherent capability of <i>Agrobacterium rhizogenes</i> to transfer root-inducing (Ri) plasmid T-DNA into the host plant genome, resulting in the formation of genetically stable, fast-growing, and hormone-independent hairy roots. Transformed hairy roots are particularly advantageous due to their high biosynthetic potential, genetic stability, and competence to replicate or even surpass the parent plant in producing bioactive metabolites such as phenolics, alkaloids, terpenoids, and flavonoids. This approach not only conserves plant biodiversity but also enables controlled manipulation of biosynthetic pathways through elicitor treatments, precursor feeding, and metabolic engineering. The integration of advanced technologies including transcriptomics, proteomics, and metabolomics further enhances our understanding of metabolic regulation and supports pathway optimization for improved metabolite yield. Despite its tremendous potential, challenges remain in optimizing transformation protocols, strain selection, and scaling up production using bioreactor systems. Nevertheless, hairy root cultures constitute an efficient and environmentally sustainable platform for the large-scale production of high-value metabolites across diverse plant species. This review explores the biological basis, influencing factors, and applications of hairy root cultures, emphasizing their role in modern plant biotechnology and their commercial relevance in the pharmaceutical and nutraceutical industries. We also critically discuss and analyze recent trends and future perspectives.</p>