In this study, the medium composition, carbon source, non-natural amino acid concentration, and induction condition were optimized for the production of recombinant urate oxidase with non-natural amino acids. Recombinant Escherichia coli strain was first cultured in various media including LB, 2 × YT and B, and cell growth and urate oxidase expression were compared. Based on the selected B medium, the concentrations of each component in medium were optimized through response surface methodology. At the optimal medium compositions, urate oxidase with non-natural amino acids was produced with 64.92 U/mg specific protein activity in flask culture. Among various carbon sources, the highest urate oxidase activity was obtained at the glucose concentration of 5 g/L. The optimal inducer concentrations were 1 mM of IPTG. 1 mM of non-natural amino acids was efficient for urate oxidase production. This results will be useful for the production of therapeutic proteins with prolonged activity