Glucose oxidase (GOx) catalyzes the oxidation of D -glucose to D -glucono-1,5-lactone and has a wide range of applications in various industries. However, the strict substrate specificity of GOx hampers its application in the conversion of other abundant sugars such as D -xylose. In this study, the substrate preference of GOx from Aspergillus niger (AnGOx) was engineered using a semi-rational design approach. The mutant T110V/F414L exhibited a 5.7-fold increase in D -xylose oxidation activity compared to that of the wild-type enzyme, which was attributed to its enhanced affinity for the substrate. Molecular dynamics simulations indicated that the T110V and F414L mutations may mitigate the non-productive binding of D -xylose at the entrance of the substrate-binding pocket, and therefore, are beneficial for providing access of its C1 hydroxyl group to the catalytic residues. Moreover, the mutant simultaneously oxidized D -xylose and D -glucose in the corncob hydrolysate to the corresponding aldonic acids when coupled with catalase. These findings provide new insights into substrate recognition by GOx and offer a new method for the utilization of D -xylose from lignocellulosic feedstocks. [ABSTRACT FROM AUTHOR]