Enhancement of secondary metabolites by means of hairy root culture is a potential method for mass production of desired metabolites from in vitro plants. Accordingly, the aim of this investigation deals with the enhancement of aervine content through hairy root cultures from in vivo leaf, callus and in vivo node of Aerva lanata. The experimentation was carried out with 24 variants of hormonal combinations 6-benzylaminopurine (BAP), 2,4-dichlorophenoxy acetic acid (2,4D), α-naphthalene acetic acid (NAA) and spermidine (SPM) for the induction of callus. The most intense growth of callus was found on media augmented with 2.0 mg/L BAP +1.0 mg/L 2,4-D as well as on media containing 1.5 mg/L 2,4-D +1.0 mg/L BAP. The Agrobacterium rhizogenes-mediated Ri transformation (MTCC 532 strain) let to the development of numerous hairy roots when treated with in vivo grown leaf. The callus when infected with MTCC 532 strain for 20 min and co-cultivated for 24 h on MS medium exhibited the maximum percentage of hairy root response per explant (80.96 ± 0.06). The integration of foreign genes was confirmed by early β-glucuronidase (GUS) staining assay followed by polymerase chain reaction (PCR) analysis based on the amplification of rolB, rolC and virD genes. The least concentrations of methanolic extract from hairy root cultures showed promising antioxidant activities. The occurrence of high peak values of hairy root extract was confirmed using high performance liquid chromatography (HPLC) which indicates the presence of aervine (alkaloid compound) in comparison with a standard. The final result indicates that hairy root culture produces enhanced aervine content, which shall be used for commercial drug production in food and pharmaceutical companies. As far as we are aware, this is the first and foremost report describing the aervine production through hairy root culture from A. lanata. © 2020 Elsevier B.V.