L-Glutaminase majorly produced by micro organism including bacteria, yeast and fungi. L-Glutaminase mainly catalyzes the hydrolysis of γ-amido bond of L-Glutamine. In this report, Optimization of the culture medium for LGlutaminase production using Streptomyces griseus was carried out. The optimization of L-Glutaminase production using Rice bran as substrate was performed with statistical methodology based on experimental designs. The screening of ten nutrients for their influence with Rice bran on L-Glutaminase production is achieved using Plackett-Burman design. The basal medium contained Peptone 30 g/L, Ferrous sulphate 0.7 g/L, KH2PO4 0.7 g/L, NaCl 40 g/L was selected based on their higher influence on L-Glutaminase production. After medium components optimization, the Temperature, pH, Time, concentration of the Rice bran, and Inoculum size was optimized using Response Surface Methodology (RSM). The predicted optimum levels are as follows: Temperature 30.60 0C, pH 8.26, Time 111.34 h, Rice bran 33.12 g/L and Inoculum size 0.906 %. This medium components and parameters were projected theoretically to produce an L-Glutaminase activity of 179.92 IU/ml. The used methodology was validated using this optimized media components and parameters; the L-Glutaminase activity 171.01 IU/ml was obtained.