In this paper, a simple and reliable fabrication method about electrochemically reduced graphene oxide (ERGNO)-prussian blue (PB) nanocomposite was proposed for determination of guanine. Due to its unique structural, physical and chemical properties, ERGNO, which was fabricated on the carbon paste electrode (CPE) beforehand through electrochemical reduction of graphene oxide, was selected as a compatible precursor for next-step PB electrodeposition. Electrochemical behaviors of the resulted PB/ERGNO/CPE were investigated by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The electrochemical results showed that PB/ERGNO/CPE exhibited good electrochemical performances. The electrocatalytic results of guanine further illustrated that graphene prompted the electrocatalytic ability of PB via the redox shift between PB and prussian yellow (PY) in the potential range from 0.5 to 1.2 V, which has not been widely adopted in the PB based electrochemical sensors. The detection limit of guanine could be calculated to be 1.0×10−8 mol/L. It means this PB/ERGNO/CPE platform is quite sensitive and can be readily applied in biosensor field.