The present work focused on the development of new simple method for fabrication of conducting poly(4-aminothiophenol) nanostructures layered on gold nanodots patterned indium tin oxide (ITO) electrode based on the self assembly of the monomer. This was followed by electrochemical polymerization of 4-aminothiophenol molecules. In addition, we studied the electrochemical catalytic activity of the modified electrode towards a mixture of two protein bases (Adenine and Guanine). The modified gold nanodots ITO electrode was fabricated based on thermal evaporation of pure gold metal onto ITO surface through polystyrene monolayer. Then, a monolayer of 4-aminothiophenol was self-assembly immobilized onto the gold nanodots array/ITO electrode. This was followed by electrochemical polymerization process based on cyclic voltammetry technique. The electrochemical behavior of the adenine and guanine mixture at the modified electrode was investigated based on differential pulse voltammetry technique. The results indicated that the polymer nanostructures modified gold nanodots/ITO electrode exhibited an excellent electrocatalytic activity towards the oxidation of adenine and guanine with a detection limit of 500 and 250 nM, respectively. Moreover, our finding demonstrated a linear relation between the concentration of both adenine and guanine and their oxidation current peaks (R = 0.9953 and 0.9935, respectively). Finally, the modified electrode was successfully used to detect adenine and guanine in human serum sample. Therefore, we proposed that this biosensor could have high sensitivity for simultaneous determination of adenine and guanine in the related physiology process. (C) 2014 Elsevier Ltd. All rights reserved.