L-asparaginase is used as a pivotal agent in treatment of lympho proliferative disorders and holds an excessive demand for food processing aids. Most of the L-asparaginases possess glutaminase activity which might lead to side effects during the treatment. The search for new therapeutic enzymes is of great interest in both medical and food applications. In this study, Vibrio cholerae L-asparaginase was recombinantly overexpressed in E. coli, purified to homogeneity and molecular size was estimated to be 36.6 kDa. Recombinant enzyme showed an optimum pH and temperature of 7.0 and 37 °C respectively. The Km and Vmax of the enzyme for L-asparagine was 1.1 mM and 1006 μM/min respectively. The enzyme is highly specific for L-asparagine and did not show glutaminase activity. The enzyme possessed high half life time and good stability over a wide range of physiological conditions. Tm of the enzyme was found to be 81 °C by circular dichroism and differential scanning calorimetric techniques. This enzyme possesses novel properties such as (i) glutaminase free L-asparaginase activity, (ii) very high Vmax value with high affinity and (iii) high stability at 30 °C (half-life time of 2.9 days). © 2017