We report the controllable fabrication of defective single-layer graphene and the investigations on kinetic rates (k(0)) of heterogeneous electron transfer (HET). Electron beam lithography (EBL) and Ar+ plasma treatment were employed to fabricate graphene patterns with different defect density. Raman spectroscopy, scanning electrochemical microscopy (SECM) and finite element simulations were performed to correlate the HET kinetic rate to the defect density (n(D)) and defect distance (L-D) of single-layer graphene. The results showed that k(0) is linearly increased with n(D) initially and then increased rapidly with n(D), which would be attributed to the density dependent interactions between defects. The facilitated HET results from the increased electronic density of states near the Dirac point of graphene, and the enlarged overlap of electronic states between graphene and redox. Increasing the defect density larger than 7.73x10(12) cm(-2) (L-D < 2.03 nm), k(0) was rapidly decreased because the disordered structures of defects decrease the conductivity. The optimal n(D) (7.17 x 10(12) cm(-2)) and LD (2.11 nm) were obtained, which leaded to 60-fold enhancement of k(0). SECM imaging combined with Raman imaging provides a powerful method for the combinatorial screening and defect engineering of other 2D materials to realize the optimization of electrochemical activity.