L-asparaginase is an anticancer agent, especially for acute lymphoblastic leukemia. Nine E.coli strains were screened forits ability to produce an extracellular L-asparaginase enzyme. The optimum culture conditions for L-asparaginase production was found at pH 8.0, Incubation time 48 h, and 37oC temperature. Highest yields of L-asparaginase(140.5 and 96.4 IU/ml) by E.coli strains using glucose and beef extract as sole carbon and nitrogen sources respectively. 0.6 fold of higher L-asparaginase activity (168.4 IU/ml) was recorded using pUC18 UV60 than the parent strain. 0.54 fold increased L-asparaginase activity (220.6 IU/ml)was observed using pUC18 NTG90.The molecular mass of L-asparaginase was determined by SDS-PAGE and it was found to be 29KDa.