An effort has been made to find the best medium and operating conditions for the production of extracellular L-asparaginase by Aspergillus terreus in shake culture fermentation through stepwise optimization strategies like studies on the effect of pH and temperature on L-asparaginase, studies on the enzyme activity in various concentration of L-glutamine and study the growth kinetics for L-glutamine medium. Our study shows that L-glutamine combined with L-asparagine influence the asparaginase production level and physical parameter such as pH and temperature has more contribution in enzyme production. The pH 8 and temperature 35°C found to be good environmental condition the production of extracellular L-asparaginase by Aspergillus terreus.