L-Asparaginase (E.C. 3.5.1.1) is the enzyme with anti-tumour activity and is well accepted as a chemotherapeutic agent against the acute lymphoblastic leukemia and lymphosarcoma. Present investigation revealed the application of three phase partitioning (TPP) along with Taguchi design of experiment to easier and scalable method for the enhanced purification of recombinant L-asparaginase. The recombinant L-asparaginase enzyme was produced by the over-expression of ansB gene of E. coli K-12 in E. coli BL21. The purification of enzyme from crude extract was carried out by TPP method. The purification level was further enhanced by using Taguchi design of experiment orthogonal array. The purification of recombinant enzyme under optimized condition has enhanced 67.26% yield with fold purification of 7.33. The maximum activity of 208.5 U and specific activity of 49.64 U/mg was identified in interfacial precipitate after the second round of partitioning. Three phase partitioning is an economical and easily scalable method for the purification of enzyme. The recombinant L-asparaginase was purified to higher homogeneity by this method. This method may be helpful in the overcoming the various problems associated with the cumbersome downstream processing.