A nonenzymatic glucose biosensor based on Pd 1 Pt 3 –graphene nanomaterials was fabricated. The Pd 1 Pt 3 –graphene nanomaterials were prepared by a one-pot microwave heating method. Atomic force microscopy, transmission electron microscopy, scanning electron microscopy, energy-dispersive spectroscopy, X-ray powder diffraction, and X-ray photoelectron spectroscopy were used to characterize the resulted nanomaterials. The electrocatalytic characteristics of the Pd 1 Pt 3 –graphene nanomaterials were evaluated for the nonenzymatic oxidation of glucose by using voltammetry. Compared with the unsupported Pd 1 Pt 3 nanomaterials, Pd 1 Pt 3 –graphene nanomaterials exhibited a great enhancement toward the oxidation of glucose. A nonenzymatic amperometric glucose biosensor with rapid response was developed with using the Pd 1 Pt 3 –graphene as an electrocatalyst in the neutral phosphate buffer solution (PBS, pH 7.4). In addition, the common interfering species, such as ascorbic acid, uric acid, and 3,4-dihydroxyphenylacetic acid did not cause obvious interference. The biosensor can also be used for quantification of the concentration of glucose in real clinical samples.