Abstract: Glucose oxidase is a group of enzymes that can catalyze β-D-glucose to produce glucono-δ-lactone, and has broad application prospects in the production of food, medicine, and biosensors. In this study, the coding sequence of glucose oxidase was cloned from Aspergillus niger, and recombined to the constitutive expression vectors pGAPZαA. The recombined plasmid was linearized by restriction endonuclease BspH1 and transformed to Pichia patoris GS115 strain using a MicroPulser electroporator. The Pichia pastoris transformants were cultivated in liquid PDA medium, and the enzymatic properties of recombinant glucose oxidase were analyzed. The results showed that the coding gene of Aspergillus niger glucose oxidase was 1 818 bp, encoding 605 amino acids with a signal peptide composed of 21 amino acids. The enzyme activity of glucose oxidase in the fermentation supernatant of Pichia pastoris constitutively expressing glucose oxidase can reach 1.2 U/mL. The purified glucose oxidase, which is about 100 kDa, was obtained by nickel ion affinity chromatography. After removing glycosylation of glucose oxidase by endoglycosidase H (Endo H), its molecular weight was about 70 kDa, which was similar to its estimated molecular weight. The study of its enzymatic properties showed that the optimal reaction pH value of the enzyme was 6.0, the optimal reaction temperature was 35℃, and the specific activity was 60.9 U/mg. To further increase the expression level of glucose oxidase in Pichia pastoris, the coding sequence of glucose oxidase from Aspergillus niger was recombined to the inducible expression vectors pPIC9K. The recombined plasmid was linearized by restriction endonuclease Pme1 and transformed to Pichia patoris GS115 strain. The inducible expression of Pichia pastoris strain was cultivated and screened through G418 antibiotic, and a highly expressing strain that can grow on the YPD agar plate containing 4 mg/mL G418 antibiotic was obtained. The high-density fermentation of this strain (200 g wet cells/L) showed that the optimal methanol addition amount was 2.5%, and the fermentation capacity of glucose oxidase could reach 133 U/mL by the 7th day of fermentation, and the protein concentration in fermentation broth was about 1.9 g/L. This study provides experimental basis for the scale-up production of Aspergillus niger glucose oxidase.