The production and extraction of enzyme L-aspiraginase produced by some fungus isolates obtained from different soil samples collected from Makkah region, in Kingdom of Saudi Arabia. There were 20 fungal isolates that were recovered from plant rhizosphere in the south and north of Jeddah city, out of which 8 isolates were able to produce L-asparaginase enzyme in high amounts. The higher producers fungal isolates were identified by using DNA sequencing of ITS-1 and ITS-4 primers and deposited in the GenBank nucleotide sequence database under accession numbers as Nothophomagossy piicola LT592943.1, Aspergillus oryzae XR002735719.1, Mucor circinelloides MF356573.1, Aspergillus oryzae HM064501.1, Aspergillus niger DQ915806.1, Penicillium sp. KP872507.1, Penicillium sp. KP256500.1, Actinomucor elegansJN887460.1, and Rhizopus oryzae GU126375.1. Nesslerization test showed the difference between the quantities produced by the strains and it was found that the maximum activity of L-asparaginase on the 5th day, was by the isolate AAB1 (1,99 U/min/ml), followed by AGL3(1.94 U/min/ml) and AGL1(1.90 U/min/ml) after six and five days of incubation, respectively.