Camptothecin (CPT), a well-known terpene indole alkaloid (TIA), exhibits anti-cancer activity by inhibiting the activity of eukaryotic topoisomerase I. The reliance on camptothecin (CPT) extraction from medicinal plants limites pharmaceutical manufacturing output. In this study, an endophytic fungus producing CPT was isolated from the seeds of Camptotheca acuminata and identified as Aspergillus tennesseensis based on morphological characteristics and molecular sequence. The CPT production was identified and quantified by High Performance Liquid Chromatography (HPLC) and High Performance Liquid Chromatography coupled to mass spectrometry (LC/MS). The intermediates of the CPT pathway, including strictosidine and strictosidinic acid, were identified using Ultra Performance Liquid Chromatography coupled to quadrupole time-of-flight electrospray ionization mass spectrometer (UPLC-QTOF-MS). These findings suggested that the endophytic fungus A. tennesseensis might harbor two CPT pathways that complemented C. acuminate via the central intermediate of strictosidinic acid. Notably, A. tennesseensis exhibited strong biotransformation, preferentially producing 10-hydroxycamptothecin over 9-hydroxycamptothecin. In C. acuminate, 10-hydroxycamptothecin increased by 224 % on the 4th day after feeding with endophyte A. tennesseensis. Treatment with 1 % methanol significantly enhanced CPT production of A. tennesseensis, resulting in a 9-fold increase (48.9 ng/g to 441.2 ng/g) in mycelium and 14.5-fold increase (0.9 μg/L to 13.4 μg/L) in culture medium, respectively. The finding of the complete CPT pathway intermediates provides us novel insights into fungal-derived camptothecin biosynthesis. The discovery of high efficient hydroxylation and methanol elicitor reveals the potential of A. tennesseensis as an alternative CPT source.