A method of aptamer-based detection of kanamycin was established by using a graphene modified glassy carbon electrode.Kanamycin specific aptamer(Kana-aptamer) could be adsorbed onto the graphene(Gr) modified electrode,which prevents the electron transfer between the probe［Fe（CN）6］3-/4- and the electrode.However,after incubation with samples containing kanamycin,kanamycin could bind with aptamer and replace it from the electrode,which in turn declines the resistance of the electron transfer and recovers the electrochemical signal.The procedure was firstly characterized by cyclic voltametry（CV） and atomic force microscopy.Then this replacement effect was utilized-to-electrochemically determine kanamycin.Under the optimized conditions,kanamycin could be determined by differential pulse voltammetry（DPV）,and the calibration curve was linear in the range of 1×10-6-1×10-5 mol/L,with a detection limit of 5×10-7 mol/L.The method was successfully applied in the detection of kanamycin in milk samples with satisfactory result.