Background: Acute lymphoblastic leukemia represents a great crusader of death for children globally. Aspergillus niger is the source of the L-asparaginase enzyme from various soil environments. Similar to the human L-asparaginase enzyme. L-asparaginase enzyme in bacteria, commonly produced by Erwinia chrysanthemum or Escherichia coli, is currently used for acute lymphoblastic leukemia management and to reduce carcinogenic acrylamide creation during food preparation. Study Objectives: Manufacture of fungal L-asparaginase enzyme as an antineoplastic factor. Methodology: In the current study, fungal L-asparaginase was produced on mineral asparagine agar (MAM) selective medium (only fungi that utilize asparagine as a sole source for carbon and nitrogen can grow on it) at PH 6.5, temperature 25⁰C, and incubation for 3 days. Malt agar medium was used for sub-culturing fungal L-asparaginase-producing strains. The biological activity and cytotoxicity activity were obstinate through nessleriazation and MTT[(dimethylthiazol-2-yl) diphenyl tetrazonium] assays respectively. Results: L-asparaginase in fungi showed high potency and bioavailability as an anticancer agent. According to morphological and biochemical tests and DNA blot hybridization, the major fungal isolate producing this enzyme was Aspergillus niger. The molecular mass was 65 KDa as discovered aside from a mass spectrometer. Vmax of 163.8 UML^{− 1} min^{− 1} and a Km value of 3.41 × 10^{− 3} M were displayed via L-asparaginase. Conclusion: This was a promising approach study and fungal L-asparaginase was generated without hypersensitivity reactions and glutaminase-like effects with superior bioactivity to bacteria.