Establishment of a hairy root culture system from the medicine plants transformed by the Agrobacterium rhizogenes has unique advantages in large scale production of secondary metabolites and provides an effective solution to the shortage of resources. Meanwhile, root-induced(Ri) plasmid is also an ideal vector for plant genetic engineering. Insect-resistant, disease-resistant genes or genes encoding key enzymes involved in biosynthesis of plant secondary metabolites can be harbored by Ri plasmid and integrated into the host plant genome for expression, thus improving the plant traits and enhancing the secondary metabolite content. Establishment of plant transgenic hairy root culture system has laid a solid foundation for regulating plant secondary metabolites content and for industrial production of pharmaceutical active ingredients by using of genetic metabolic engineering strategies. This paper reviews the latest research advances in this field and the related applications.