The effect of defective graphene flake (DGF) as supports for Pd nanocubes (Pd NCs) in the catalysis of D-glucose oxidation reaction (GOR) was studied. The DGF used to support the Pd NCs were prepared via both a sonoelectrochemical method (DGF SECM ) and a chemical reduction method (DGF CM ). The comparable data supported by the diffraction electron pattern of transmission electron microscope (TEM) and X-ray diffraction (XRD) spectra evidence that the corners of the Pd NCs faced up toward the both DGF supports, leading to an increase in the XRD intensity of the Pd (111) peak. Additionally, a fair electrochemical comparison of the DGF support effect on GOR catalysis via Pd NCs in a NaOH electrolyte indicated that the DGF SECM -supported Pd NCs (Pd NC/DGF SECM ) and DGF CM -supported Pd NCs (Pd NC/DGF CM ) demonstrated earlier onset potentials and an overall order of Pd NC/DGF SECM > Pd NC/DGF CM > commercial Pd/C catalyst > Pd NCs for the peak charge accompanying the formation of glucolactone was obtained. Through the use of a DGF SECM substrate, the electrochemical surface area and charge transfer resistance toward GOR were significantly improved. In addition, as supported by the catalytic activity for GOR, Pd NC/DGF SECM showed remarkable sensitivity and tolerance to foreign substances in the application as a non-enzymatic glucose sensor, where Pd NC/DGF SECM showed higher two-period sensitivities of 74 and 45.86 μA · mM −1 · cm −2 for 0.25-5 and 5-24 mM D-glucose, respectively. The high recovery in serum sample analyses further confirmed the potential of Pd NC/DGF SECM as glucose sensors.