The electrochemical behavior of guanine and adenine on the graphene and Nafion composite film modified glassy carbon electrode was investigated by differential pulse voltammetry (DPV). The results indicated that the modified electrode exhibited an excellent electrocatalytic activity towards the oxidation of guanine and adenine, testified by the increased oxidation peak current and decreased oxidation potential. The experimental conditions were optimized. The separation of the two oxidation peaks was 0.364 V in 0.1 M pH 4.4 acetate buffer solution (ABS). Based on this, a novel electrochemical method was proposed to simultaneously cletermine guanine and adenine with the detection limit of 0.58 (guanine) and 0.75 (adenine) mu M (S/N = 3). The proposed method was applied to determine guanine and adenine in milk powder, urine and herring sperm DNA samples with satisfactory results. The value of (G + C)/(A +T) in herring sperm DNA was calculated to be 0.8065. The fabricated electrode showed excellent reproducibility, stability and anti-interference. (C) 2010 Elsevier Ltd. All rights reserved.