Cancer treatments continue to have many disadvantages. Reactive oxygen species, such as H2O2, in high concentrations, can cause cytotoxicity to cells, being even greater in cancer cells. One of the H2O2-producing enzymes is glucose oxidase; its application in cancer treatment should be explored. In this work, the extracellular expression of the mutated recombinant enzyme glucose oxidase was carried out in the eukaryotic expression system Pichia pastoris SMD1168, through the modification and optimization of the gox gene of Aspergillus niger to improve its expression in yeast and its purification. Also, the secretion signal of the alpha-mating factor from Saccharomyces cerevisiae was added to the gene for extracellular expression, and it was inserted into the expression vector pPIC3.5k. The extracellular expression of the enzyme facilitated purification by anion exchange chromatography; the purification was corroborated by SDS-PAGE, with a molecular weight of its subunit between 63 kDa and 100 kDa. The mutated recombinant enzyme glucose oxidase showed greater anticancer activity compared to the commercial glucose oxidase and could have potential for cancer treatment. KEY POINTS: • Pichia pastoris is an excellent eukaryotic expression system for proteins that need post-translational modifications. • Extracellular expression facilitates protein purification. • Glucose oxidase has potential application in cancer treatment.