To demonstrate the role of glucose oxidase (GOD) on the enzyme-catalyzed production of xylose from SCB hemicellulose, a variant of the GOD gene, which contains two amino acid substitutions, was cloned from Aspergillus niger CICC40179. Three-dimension structure modeling showed that the two variable amino acid residues were positioned on the surface of the protein molecule. The GOD gene was expressed in Pichia pastoris. Approximately 16.31 U/mL of GOD was obtained after 3 days of fermentation in the optimized medium. The catalytic characteristic of GOD was considered to apply in the production of xylose from hemicellulose. The pH value, temperature and the loading of GOD were optimized using the Box–Behnken design to improve the yield of xylose. The xylose yield was increased by 46% in 16 h of hydrolysis by adding GOD when the reaction was conducted under the optimal conditions (pH 5.1, temperature 44.9 °C and 31.68 U GOD per gram substrate). Glucose was not detected in the hydrolysis system supplemented with GOD. This is the first study demonstrating the application of glucose oxidase (GOD) on the enzyme-catalyzed production of xylose from SCB hemicellulose. The newly developed enzymatic hydrolysis system can contribute to the industrial production of xylose.