L-Glutaminase is widely distributed in microorganisms including bacteria, yeast and fungi. The enzyme mainly catalyzes the hydrolysis of ?-amido bond of l-glutamine. In this report medium optimization was conducted through one -factor -at -a -time approach for the submerged production of L-Glutaminase by Streptomyces griseus using different additional carbon, nitrogen, amino acids, mineral salts and was treated with different concentration sodium chloride. A significant influence of medium components (g/l) Galactose 10.0,Yeast extract 10.0,L-Glutamine 10.0,Magnesium sulphate 0.5, KH2PO4 0.5 , K2HPO4 0.5, NaCl 40 on L-Glutaminase production was noted. The applied methodology was validated using this optimized media, the enzyme activity 45 IU/ml in 48h of incubation was obtained.