L-asparaginase is known to be the most hopeful and upcoming anti-tumour enzyme helpful in reduction of the level of amino acid L-asparagine resulting in cancer cell starvation and leading to the cell death. Although, there are many sources of asparaginase enzyme yet bacterial source i.e. E.coli and Erwinia species is mostly used as a therapeutic agent against leukaemia. In presented study screening of various bacteria is carried out by rapid plate assay for asparaginase. Enzyme producing then intracellular and extracellular Asparaginase were extracted from its potent producer i.e. E.coli that is isolated from sewage water. Obtained results showed that an intracellular asparaginase enzyme is found to be more active against asparagines as compare to the extracellular enzyme.