The enzyme Asparaginase, known as L-asparagine amidohydrolase, has anti-tumor properties and is widely used as a chemotherapeutic treatment for acute lymphoblastic leukemia. Many different organisms, including plants and terrestrial and microbes such as bacteria, fungi, algae and actinomycetes, have produced this enzyme. In this study, 34 isolates of the actinomycetes were collected from soil and water from different governorates in Egypt. Only nine Streptomyces isolates were identified by the presence of a pink hue around their colonial growth as an indication and had the best ability for the synthesis of extracellular Asparaginase.  Glucose and peptone were effective carbon and nitrogen sources, respectively. The ideal pH for Asparaginase is 7, and the ideal temperature is 30 ⁰C. and an incubation time of 7 days with a 0.4 % asparagine concentration.