The direct electrochemistry of glucose oxidase (GOx) integrated with graphene was investigated. The voltammetric results indicated that GOx assembled on graphene retained its native structure and bioactivity, exhibited a surface-confined process, and underwent effective direct electron transfer (DET) reaction with an apparent rate constant (k s) of 2.68s−1. This work also developed a novel approach for glucose detection based on the electrocatalytic reduction of oxygen at the GOx–graphene/GC electrode. The assembled GOx could electrocatalyze the reduction of dissolved oxygen. Upon the addition of glucose, the reduction current decreased, which could be used for glucose detection with a high sensitivity (ca. 110±3μAmM−1 cm−2), a wide linear range (0.1–10mM), and a low detection limit (10±2μM). The developed approach can efficiently exclude the interference of commonly coexisting electroactive species due to the use of a low detection potential (−470mV, versus SCE). Therefore, this study has not only successfully achieved DET reaction of GOx assembled on graphene, but also established a novel approach for glucose detection and provided a general route for fabricating graphene-based biosensing platform via assembling enzymes/proteins on graphene surface.