The aim of this work was to purify L-glutaminase from Aspergillus flavus. The enzyme was purified 12.47-fold from a cell-free extract with a final specific activity of 613.3 U/mg and the yield was 51.11%. The molecular weight of the enzyme, as estimated by SDS-PAGE, was found to be 69 kDa. The maximal activity of L-glutaminase was recorded at pH 8 and 40°C. The highest activity was reported towards L-glutamine as substrate, with an apparent Km value of 4.5 mmol and Vmax was 20 Uml-1. The enzyme was activated by Na+ and Co2+, while it was greatly suppressed by iodoacetate, NEM, Zn2+ and Hg2+ at 10 mM. L-glutaminase activity increased with a gradual increase of sodium chloride concentration up to 15%. In vivo, the median lethal dose (LD50) was approximately 39.4 mg/kg body weight after intraperitoneal injection in Sprague Dawley rats. Also, L-glutaminase showed no observed changes in liver and kidney functions and hematological parameters on rates. Purified A. flavus L-glutaminase had neither a cognizable effect on human platelet aggregation nor hemolytic activity. In addition, MTT assay showed that the purified L-glutaminase has a high toxic impact on Hela and Hep G2 cell lines with an IC50 value 18 and 12 μg/ml, respectively, and a moderate cytotoxic effect on HCT-116 and MCF7 cells, with an IC50 value 44 and 58 μg/ml, respectively.