It is urgent to develop a more simple and sensitive method to detect antibiotic residues considering the harm of antibiotic residues in food to the human body. Herein we designed a label-free electrochemical DNA biosensor for the sensitive detection of kanamycin (KAN) based on diblock DNA with a 15-mer of poly-cytosine (poly-C). The diblock DNA can be immobilized on graphene oxide (GO) due to strong physical adsorption between the 15-mer of poly-C and GO. The aptamer of KAN acted as the other block for rapidly binding the target. It can specifically capture the target, which leads to the change of electrochemical signal. Consequently, the DNA biosensor exhibited high sensitivity and specificity towards KAN, the linear range was from 0.05 pM to 100 nM with a detection limit of 0.0476 pM. The developed DNA biosensor was constructed easily and showed promising applications for the detection of antibiotic residues for food safety.