Catharanthus roseus (L.) G. Don is a plant species known for its production of a variety of terpenoid indole alkaloids, many of which have pharmacological activities. Catharanthine can be chemically coupled to the abundant leaf alkaloid vindoline to form the valuable anticancer drug vinblastine. To study and extract catharanthine and other metabolites from C. roseus, a technique was developed for producing hairy root cultures. In this study, the Agrobacterium rhizogenes A4 was induced in the hairy roots from leaf explants, and the concentration of antibiotics (100 mg/L kanamycin) was elucidated for selection after transformation. The polymerase chain reaction amplification of rol genes results revealed that transgenic hairy roots contained rol genes from the root induced (Ri)-plasmid. Catharanthine from C. roseus hairy roots was separated and analyzed using high-performance liquid chromatography. Over-expression of CrOrca3 (octadecanoid-responsive Catharanthus AP2/ERF domain), and cytohistochemical staining methods were used to validate transgenic hairy roots from C. roseus. Hairy root culture of C. roseus is a valuable approach for future efforts in the metabolic engineering of terpenoid indole alkaloids in plants.