The discovery that L-asparaginase acts as an antitumor agent in children with lymphoblastic leukemia led to the extensive studies on the physicochemical properties of the enzyme as well as on clinical effect. In the present study thirty isolates of actinomycetes from Egyptian soil were screened for production of L-asparaginase enzyme using Batch fermentation. The most active producer was identified as Streptomyces halstedii. Optimal cultural factors affecting the production of L-asparaginase by Streptomyces halstedii on glycerol-asparagine broth were pH 7.0, 30 ºC for 5 days. The strain utilized glycerol and L-asparagine as the best carbon and nitrogen sources for L-asparaginase production. The enzyme was purified to homogeneity by ammonium sulfate precipitation, dialysis, and Sephadex G-200 gel filtration. The enzyme was purified to about 55.2 folds with a final specific activity of 2071.2 U/mg protein and 4.17% yield. The molecular weight of the enzyme was determined to be 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.