Background Emerging of endophytic fungi as potent camptothecin producers raise the hope for its commercial production, due to their rapid growth and feasibility of metabolic engineering, nevertheless, their loss of productivity with the fungal storage and subculturing is the challenge. Thus, screening for unique fungal isolate with sustainable camptothecin productivity is the objective of this work. Results The camptothecin productivity of the fungal endophytes of wild and in vitro cultured Astragalus fruticosus was evaluated. Aspergillus flavus ER, endophyte of A. fruticosus explant, was the potent producer (51.7 μg/l), the chemical identity of putative compound was resolved by UV, HPLC and LC-MS/MS analyses. The purified A. flavus camptothecin displayed a significant activity against HEPG-2 (IC50 0.9 mM), MCF7 and HCT29 (IC50 1.2–1.35 mM). The productivity of camptothecin by A. flavus was increased by 1.6 fold with methyljasmonate. Upon Plackett-Burman Design optimization, the yield of camptothecin was enhanced by 3 fold (150 μg/l) comparing to control. The camptothecin biosynthetic machinery of A. flavus was noticed to be attenuated with subculturing, nevertheless, this biosynthetic potency was restored upon addition of A. fruticosus methanolic extract (1%), ensuring the incidence of specific signals from plant tissues that triggers the expression of camptothecin encoding genes. Conclusion This is the first study deciphering the feasibility of A. flavus for sustainable production of camptothecin upon addition of A. fruticosus extracts, that could be a new platform for camptothecin scaling-up.