Urate oxidase (UOX) is used for the treatment of hyperuricemia and gout. The low stability of protein drugs such as UOX is a significant drawback in their liquid solutions. In this study, UOX from Aspergillus flavus was overexpressed and purified. Then, the effect of osmolytes (glycerol, sorbitol, and sucrose) on UOX thermal stability was studied. The enzyme stability was optimized by the aid of osmolytes using response surface methodology (RSM). The thermal stability results and Pareto analysis showed that sucrose has a stronger stabilization effect on UOX relative to the other osmolytes and increases the enzyme stability up to 2.4-fold, solely. RSM results indicated that the stability of UOX increased up to 4.9-fold in the presence of 12 % of glycerol and sucrose. The combinatorial effect results indicated that the osmolytes affect each other stabilization effects on UOX, probably through influencing the hydrogen bonds and mixing the osmolytes at different concentrations does not always lead to increased stability of UOX. The results revealed that the effect of sorbitol on UOX stability was negligible in the presence of glycerol and sucrose. Finally, this study results showed that RSM can be used to select a proper set of osmolytes for enzyme stabilization.