The study was conducted to show that Agrobacterium rhizogenes–mediated transformation enhances the yield of biologically active metabolites. Various explants of Aerva javanica, such as in vivo shoots, in vivo leaves, and in vitro callus, were used in the study. The callus induction of shoot and leaf explants was carried out using Murashige and Skoog (MS) medium at different concentrations of 2,4- dichlorophenoxyacetic acid (2,4- D) and α-naphthalene acetic acid (NAA) at 1.0 mg/L. The transformation was carried out using different strains of A. rhizogenes (ATCC 15834, MTCC 2364, MTCC 532, R1000, and LBA 9204). Of the five tested strains, ATCC 15834 showed the best transformation efficiency (69.67%) and higher efficiency in regenerating hairy roots (HRs). Using the GUS staining and Polymerase Chain Reaction (PCR) method, it was observed that the rol genes alone were expressed in the transformed roots. ultra-performance liquid chromatography (UPLC) revealed the enhancement of aervine in the in vitro HRs after 42 days of incubation with ½ strength liquid MS medium without auxin. HR regenerated from ATCC 15834 accumulated maximum quantity of aervine (45.21 ± 0.58 μg g−1 d.w.). An in silico model showed that aervine binds to proteins with a high affinity, indicating that the enhanced molecules could be a promising cancer treatment. Aervine enriched hairy roots from in vitro callus (AEHRIC) extract were studied with the MTT cytotoxicity assay against human (MCF-7) breast cancer line with an IC50 value of 37.18 ± 0.36 µg/ml. The final report confirmed that the HR culture enhanced the aervine content and strong enough to control breast cancer.