Abstract: Glucose oxidase (GOD) is widely used in food, chemistry, medicine, biotechnology and other industrial applications. In this study, the gene GOD from Aspergillus niger was optimized according to the codon bias of pichia pastoris(P. pastoris), then it was used to construct the GOD secretory expression vector pPIC9K-GOD and the corresponding recombinant P. pastoris strain G/GOD. Subsequently, the higher concentration of geneticin (G418) was used to select the multicopy genomic integration strain G/GODM, and the extracellular GOD specific activity was improved to 5843.2 U/g, which was 8.2 times as high as that of the single copy strain G/GOD. On the basis of the first generation high producing strain, additional co-expression of folding factors, as well as the enhancement of central carbon metabolism, were used to construct the second generation strain. Co-expression of the protein folding factors PDI1, PDI2 and HAC1 improved the extracellular GOD activity by 32.7%, 8.9% and 54.4%, respectively. With the co-expression of the pentose phosphate pathway gene SOL3 and the tricarboxylic acid cycle gene MDH1, the extracellular GOD activity was enhanced by 6.3% and 11.6%, respectively. The best strain G/GMH1 was selected for the bioreactor fermentation to achieve 6 656.6 U/g of the extracellular GOD activity in a 50 L bioreactor, indicating its value for industrial application.