A simple process in which a graphene oxide (GO)-Prussian Blue (PB)-3,4,9,10-perylenetetracarboxylic dianhydride derivative (PTC-NH2) nanocomposite film is spread onto a glassy carbon electrode (GCE) surface is described. Glucose oxidase (GOD) was adsorbed on the modified glass carbon electrode via the specific structure of hollow Pt nanospheres. This process was sufficient to prevent GOD from leaching away, and to improve the sensitivity of our sensor. To characterise the sensors, several techniques were employed, including transmission electron microscopy (TEM), Fourier transform infrared (Fr-IR) spectroscopy, cyclic voltammetry (CV), electric impedance spectroscopy (EIS) and chronoamperometry. The resulting amperometric glucose biosensor exhibited a fast response time (within 5 s) and a good linear calibration range from 0.01 +/- 0.06 mM to 5.23 +/- 0.04 mM with a lower limit of detection of 33 mu M (SIN = 3), Possible interferents, including ascorbic acid, 1-cysteine, dopamine and ethanol, showed almost negligible electrochemical responses, indicating the high specificity of the proposed biosensor for glucose detection. The improved performance of the proposed electrode for detecting glucose in human serum was ascribed to the high surface-to-volume ratio and the excellent conductivity of the hollow Pt nanospheres. This skilful strategy of incorporating the GO/PB/PTC-NH2 nanocomposite film with hollow Pt nanospheres enhances the performance of the electrochemical sensor, which holds promising for application in bioassay analysis. (C) 2015 Elsevier B.V. All rights reserved.