Abstract: A total of ten actinomycete strains were isolated from the soil sample. All the strains were sub cultured as pure on Yeast extract, Malt extract and Dextrose agar and screened for the biologically active enzymes, and optimization of L.aspariginase enzyme was carried out by various parameters including pH, temperature, and carbon, nitrogen sources by growing the strain CN-4 in modified ISP-5 broth for 96 h. Among the seven carbon sources tested, lactose proved to be the best carbon source by supporting high yields of L-asparaginase enzyme. Influence of six nitrogen sources on L-asparaginase production and growth of the strain was tested. Yeast extract was found to enhance both growth and production of L-asparaginase. Once the preferable carbon and nitrogen sources were determined as lactose and yeast extract, efforts were made to identify their suitable concentrations. Lactose @ 0.5% and yeast extract @ 0.5% with 1% L-asparagine as optimized nutrient broth adjusted to pH 7.0 inoculated with Streptomyces albus CN-4 incubated at 30°C for 96 h improved the production of L-asparaginase.