L-Asparaginase is widely used enzyme for the treatment of ALL and thus its Production and activity plays a major role in its therapeutic use. Screening of the different sources for prominent L-Asn-ase production and high activity was done at different physiological conditions and high yield of the enzyme was obtained. The obtained enzyme from the two isolates are compared relatively on the basis of Km and Vmax values and one of them is being found to be more effective with low Km. This would serve as a good drug if tested and certified clinically restricting the ALL.