A promising electrochemical microsensor was fabricated by electrochemical immobilization of DNA on a carbon fiber electrode(CFE) through an overoxidized polypyrrole(PPyox) template for simultaneous determination of serotonin(5-HT) and dopamine(DA). Such a nano-thickness DNA-PPyox biocomposite modification layer exhibited a superior selectivity and sensitivity towards these neuronal amines comparing with a simple PPyox or DNA coating. This microsensor gave three separated differential pulse voltammetric(DPV) peaks at 0.38, 0.20 and -0.02 V for 5-HT, DA and 25000-fold excess of ascorbic acid(AA). The utility of this composite biosensor was demonstrated by the determination of 5-HT content in healthy human blood serum and DA in its hydrochloric injection.