L-Asparaginase?(L-ASNase EC 3.5.1.1) is considered as an important biopharmaceutical drug enzyme in the treatment of childhood acute lymphoblastic leukemia (ALL). In the present study,?Pyrococcus?furiosus?L-ASNase gene was cloned into pET26b (+), expressed in E. coli BL21(DE3) pLysS, and purified to homogeneity using Ni2+?chelated Fast Flow Sepharose resin with 5.7?purification?fold and 23.9% recovery. The purified enzyme exhibited a molecular weight of ~33,660?Da on SDS-PAGE and showed maximal?activity?at 50?°C and pH?8.0. It retained 98.3% and 60.7% initial?activity?after 60?min at 37?°C and 50?°C, respectively. The?recombinant?enzyme showed highest substrate specificity towards L-ASNase substrate, while no detectable specificity was observed for l-glutamine, urea, and acrylamide at 10?mM concentration. The Km and Vmax of the purified?recombinant?enzyme as calculated using Lineweaver-Burk plot were determined to be 1.623?mM and 105?μmol?min-1?mg-1, respectively. Human leukemia?cell?line?THP-1?treated with?recombinant?L-ASNase showed significant morphological changes, and the IC50?of the purified enzyme was found to be 0.8?IU. Moreover, the purified?recombinant?L-ASNase induced cytotoxic effects on lung adenocarcinoma?A549?and colorectal adenocarcinoma?Caco-2?cell?lines?with IC50?of 1.78?IU and 30?IU, respectively.