A homogeneous electrochemical assay based on a graphene monolayer electrode was developed for simple, sensitive, rapid and quantitative analysis of the exonuclease III (Exo III) activity. The method utilized a methylene blue (MB) tagged DNA substrate with hairpin structure, and a graphene monolayer attached on the working electrode. Before digestion, the hairpin structure prevents the adsorption of the DNA substrate to the graphene surface. Degradation of the substrate by the 3′–5′ Exo III, however, yields single‐stranded DNA (ssDNA), resulting in its subsequent binding to the graphene surface through π‐π stacking, which produces the voltammetric current from electrochemical reduction of the MB tag anchoring at the end of ssDNA. A direct quantification of the Exo III activity can be achieved by measuring the reductive peak current of the MB tag under easily attainable potential (∼ −0.1 V vs Ag/AgCl) range comparably sensitive to the conventional methods such as a gel‐based or fluorescence‐based assays. Our approach can be applied to measure various exonucleases activity by adjusting the structure of DNA substrate suggesting a new assay method in drug screening and basic research related to the enzymes.