Hyperuricemia, caused by impaired uric acid excretion, poses significant health risks. Urate oxidase (UOX) from Aspergillus flavus offers therapeutic potential by converting uric acid into soluble allantoin; however, its instability limits clinical applications. This study investigated the effects of osmolytes, including proline and glycine, on the kinetics and thermostability of recombinant A. flavus UOX. Following the expression of UOX coding sequence in Escherichia coli BL21, it was purified using Ni2+-NTA agarose affinity chromatography and confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme maintained its activity up to 35.00 ˚ C and lost its activity at higher temperatures as it lost 70.00 % of its activity after 60 min at 40.00 ˚ C, and the enzyme with proline and glycine additives maintained 73.00% and 30.00% of the activity, respectively. The inactivation rate constant of enzyme (k_in) was decreased in the presence of proline, indicating that the enzyme was more stable with proline, but glycine had no effect on kin. Half-life of enzyme was raised to 86 min in the presence of proline and the Michaelis constant (K_m) was decreased significantly by both osmolytes, as well. These results demonstrated that proline stabilized UOX by mitigating thermal denaturation, likely through preferential hydration and hydrophobic interactions, while glycine enhanced substrate binding. The stabilizing capacity of proline highlighted its utility for inclusion in biopharmaceutical formulations, offering a solution to the persistent challenge of UOX instability in therapeutic contexts. These findings yielded practical strategies for enhancing both structural integrity and catalytic performance of enzymes in pharmaceutical development.