L-asparaginase is an extremely demanding biocatalyst that is employed to treat acute lymphoblastic leukemia (ALL) and lessen the development of acrylamide in fried food products. Here, an endophytic F. oxysporum MJS2, obtained from the stem tissues of M. jalapa was evaluated for the synthesis of L-asparaginase. The fermentation conditions were optimized through One Variable At a Time (OVAT) joined with a Central Composite Design (CCD) for the maximum production of enzymes. A 2.32-fold increase in the enzyme action was detected in the post-optimized condition (32.47 U mL-1) in a fermentation condition of pH 7, incubation temperature of 37 °C, and 120 hours of incubation time, glucose (5 gL-1), ammonium sulphate (7 gL-1), and NaCl were the best options for L-asparaginase synthesis by the endophyte. Crude enzyme was dialyzed and purified using Sephadex G-100 column chromatography with a molecular weight of 35 kDa determined through SDS-PAGE. MJS2-derived L-asparaginase acts optimum at a substrate concentration of 50 mM. Endophytic fungi MJS2 could be pharmaceutically exploited to produce L-asparaginase and open up new horizons in the biotechnological aspects of endophytes of common Indian Medicinal plants.