Understanding the heterogeneous electron-transfer (ET) kinetics on graphene is essential for its extensive applications. Here, on the basis of the redox-induced fluorescence variation of monolayer graphene itself, the heterogeneous ET kinetics at the interface between the electrode and the monolayer graphene was studied label-freely at the single-sheet level. By tuning the defect density on graphene, an optimal heterogeneous ET rate was observed at a moderate defect density, indicating defect-driven ET kinetics. The heterogeneities of both the intrasheet and intersheet ET kinetics were revealed at the single-sheet level. With the optimal defective graphene sheets as a sensing material for oxygen gas, a cost-effective electrochemical oxygen sensor was obtained with high sensitivity, fast response/recovery, and remarkable durability. The results obtained here deepen our understanding of the electrochemical properties of graphene and imply that rational defect control can enhance the ET process between the electrode and graphene and then improve the performance of graphene-based functional materials or devices.