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l-Asparaginase (l-ASNase) is a key enzyme used to treat acute lymphoblastic leukemia, a childhood blood cancer. Here, we report on the characterization of a recombinant l-ASNase (Ps44-asnII) from Pseudomonassp. PCH44. The gene was identified from its genome, cloned, and overexpressed in the host Escherichia coli(E. coli). The recombinant l-ASNase (Ps44-ASNase II) was purified with a monomer size of 37.0 kDa and a homotetrameric size of 148.0 kDa. The purified Ps44-ASNase II exhibited optimum activity of 40.84 U/mg in Tris–HCl buffer (50 mM, pH 8.5) at 45 °C for 15 min. It retained 76.53% of enzyme activity at 45 °C after 120 min of incubation. The half-life and Kdvalues were 600 min and 1.10 × 10–3min−1, respectively, at 45 °C. The kinetic constants values Kmand Vmaxwere 0.56, 0.728 mM, and 29.41, 50.12 U/mg for l-asparagine and l-glutamine, respectively. However, kcatfor l-glutamine is more (30.91 s−1) than l-asparagine (18.06 s−1), suggesting that enzymes act more efficiently on l-glutamine than l-asparagine. The docking analysis of l-asparagine and l-glutamine with active site residues of the enzyme revealed a molecular basis for high l-glutaminase (L-GLNase) activity and provided insights into the role of key amino acid residues in the preferential enzymatic activities.