An electrochemical aptasensor was developed for the detection of kanamycin based on the synergistic contributions of chitosan-gold nanoparticles (CS-AuNPs), graphene-gold nanoparticles (GR-AuNPs) and multi-walled carbon nanotubes-cobalt phthalocyanine (MWCNTs-CoPc) nanocomposites. The aptasensor was prepared by sequentially dripping CS-AuNPs, GR-AuNPs and MWCNTs-CoPc nanocomposites onto a gold electrode (GE) surface. During the above process, these nanomaterials showed a remarkable synergistic effect towards the aptasensor. CS-AuNPs, GR-AuNPs and MWCNTs-CoPc as the nanocomposites mediator improved electron relay during the entire electron transfer process and the aptasensor response speed. The electrochemical properties of the modified processes were characterized by cyclic voltammetry (CV). The morphologies of the nanocomposites were characterized by scanning electron microscopy (SEM). The experimental conditions such as the concentration of the aptamer, the time, temperature and the pH were optimized. Based on the synergistic contributions of CS-AuNPs, GR-AuNPs and MWCNTs-CoPc nanocomposites, the proposed aptasensor displayed high sensitivity, high specificity, a low detection limit (5.8 × 10(-9) M) (S/N = 3) and excellent stability. It was successfully applied to the detection of kanamycin in real milk spiked samples.