A highly effective Agrobacterium-mediated transformation approach was availed to enhance the production of bioactive metabolites in Aerva javanica. This study reported that callus-based transformation exhibited high efficiency and best reproducibility. The optimized hairy root inducing media devoid of plant growth regulators and strain ATCC15834 induced the hairy root (69.67%) from callus with multiple roots and high efficiency. The factors influencing the rate of callus transformation are as follows; different strains of Agrobacterium rhizogenes (ATCC 15834, MTCC 2364, MTCC 532, R1000, and LBA 9204), cocultivation time of 2 days, and infection time period of 20 min. The stable transformation by the influence of rol genes was confirmed by polymerase chain reaction. The ultra-performance liquid chromatographic analysis quantified the yield of aervine (81.41 ± 2.82 µg/mL) in aervine enriched chloroform hairy root extract from in vitro callus (AECHRIC). Initial in silico analysis for aervine on breast cancer target proteins produced low-binding energy against PR (−8.1 kcal/mol) and AECHRIC showed the better cytotoxic effect on MCF-7 (149.86 ± 0.32 µg/mL) cell line. Due to the dramatic effect of AECHRIC, it could be considered as a potent anticancer drug in the pharmaceutical hub.