An electrochemically pretreated pencil graphite electrode (PGE) has been proposed for simultaneous determination of dopamine (DA) and uric acid (UA) in the presence of high levels of ascorbic acid (AA). The electrochemical pretreatment of PGE was achieved in phosphate buffer (pH 7) by the potential recycling between 1.5-2.0 V/SCE for 100 cycles, and at the scan rate of 100 mV s(-1). The influence of electrochemical pretreatment of PGE on the separation of overlapped peaks of AA, DA and UA were studied and optimum conditions were suggested. Differential pulse voltammetry (DPV) was used for simultaneous determination of DA and UA in the presence of high concentrations of AA. The peak separation between DA and AA, and also between DA and UA was up to 260 and 160 mV, respectively. The calibration curves were in the range of 0.15-15 mu M and 0.3-150 mu M for DA and UA in the presence of AA, respectively. The detection limits (S/N =3) were 0.033 and 0.12 mu M for DA and UA, respectively. The results reveal that pretreatment of the pencil graphite electrode surface not only improves its electrochemical catalytic activities towards the oxidation of DA and UA, but also resolves the overlapped oxidation peaks of AA, DA and UA into three well-defined peaks. The present method can apply for UA and DA determination in human serum and urine, and also for some commercial pharmaceutical samples without additional sample pre-treatment. (C) 2012 Elsevier Ltd. All rights reserved.