Abstract : Actinomycetes were isolated from different estuarine sediments in the regions of Bay of Bengal off shores from Parawada to Visakhapatnam, India for extracellular Lasparaginase production. 5 different samples, 20 Actinomycetes strains were isolated. Among them 5 strains showing pink colour zones with high intensity were selected for the production of L-asparaginase. Out of 5 Actinomycetes, Strain PW-A12 shows maximum L-asparaginase activity with a yield of 3.933 IU/ml for 24 hrs Submerged Fermentation time. Further, optimizations of parameters were conducted with the strain PW-A12, by consecutive evaluation method. Maximum enzyme production with a yield of 5.73 IU/ml was observed with 4% peptone at pH 7.5. Reduced enzyme production was observed (5.6 IU/ml) while using on Yeast extract concentration of 1.2 gm /100 ml (w/v) and at a pH of 7.0 found to be optimal culture condition by the strain Streptomycetes species PW-A12 by submerged fermentation Process. Hence the present study indicated a scope for exploring estuarine Actinomycetes as a source for extracellular L-asparaginase production.