Recombinant A. flavus Urate Oxidase (rUOX), which catalyzes uric acid to allantoin, is highly effective in treating tumor lysis syndrome (TLS). However, its production process is quite complicated, and its use as a therapy is often prohibitive because of the high cost. In this work, we demonstrate, for the first time, a new bioprocess for the production of rUOX expressed in P. pastoris GS115 intracellularly in a soluble and active form, which aims to develop biosimilars that address the current limitations. For the downstream process, the homogenate clarification and intermediate purification were rationally designed with regards to protein characteristics, including polyethyleneimine precipitation, ammonium sulfate precipitation, and flow-through module anion-exchange chromatography. Response surface methodology (RSM) was conducted to optimize the cation exchanger polishing step to determine the appropriate conditions that satisfy the purity and yield requirements. Finally, 2.2 g rUOX (99% purity) was obtained from the 1 l fermentation broth. Various characterizations were carried out to confirm rUOX's similarity to its commercial counterpart with regards to both physicochemical and enzymatic aspects. Overall, the rUOX derived from P. pastoris has the potential to be used in therapeutic applications.