Abstract: L-asparaginase can be effectively used for the treatment of acute lymphoblastic leukemia and tumour cells. For pharmacological and clinical tests, microbial sources are best for the bulk production of L-asparaginase. A reported work is mainly focus on the production of L-asparaginase from coliform bacteria as they are involved in an elaboration of L-asparaginase. Coliform bacteria were isolated from the water samples which were collected from the various places of Belgaum by using modified ISP-5 medium and multiple tubes lactose fermentation method. Of the 12 isolates AS-2 strain was found to be the Citrobacter sp. and also involved in an elaboration of L-asparaginase. It was characterized by taxonomical and morphological studies. The effect of carbon and nitrogen sources on the production of L-asparaginase was investigated. Glucose at 1.0% and Yeast extract at 1.5% exhibited maximum L-asparaginase production. The other parameters such as pH, temperature and incubation period were optimized and set at 8, 37°C and 26 h, respectively. L-asparaginase has been partially purified with ammonium sulphate (20-40%).