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.