L-Glutaminase, an amidohydrolase enzyme has been a choice of interest in the treatment of lymphoblastic leukemia. This study investigates the production and optimization of extracellular glutaminase enzyme using several agro-industrial residues by Aspergillus flavus KUGF009 using SSF (solid state fermentation). Effect of process variables namely substrates, incubation period, temperature, moisture content, initial pH, supplementary carbon and nitrogen sources and metal ions on the production of L-glutaminase was studied and accordingly, optimum conditions were determined. A. flavus KUGF009 was cultured in tea dust to produce L-glutaminase. The organism produced high levels of glutaminase under optimized culture conditions on the 5th day of incubation at an optimum pH 4.0, temperature 30°C and moisture content 50% by SSF. Enhanced production occurred on the addition of dextrose, yeast extract and MgSO4 as nutritional factors.Key words: L-Glutaminase, anti-leukemic agent, Aspergillus flavus, solid state fermentation