A novel bio-composite film which contains multi-walled carbon nanotube (MWNT)-chitosan (Chit)/poly(amidoamine) (PAMAM) nanocomposite along with the incorporation of DNA modified Au electrode as a biosensor for determination of dopamine(DA) and uric acid (UA) under coexistence of ascorbic acid (AA) was fabricated by layer-by-layer modification. Electrochemical impedance spectroscopy (EIS) and Cyclic Voltametry (CV) were used to characterize the electrochemical properties of the modified electrodes. The biosensor was applied to detect DA and UA in the presence of M. It not only exhibited strong catalytic activity toward the oxidation of DA and UA but also separated the originally overlapped signals of UA, DA and AA oxidation at the bare electrode into three well-defined peaks. The peak separation between M and DA, AA and UA was 179 mV and 288 mV, respectively. In the presence of 1.0 mM AA, two linear relationships were obtained for DA over the concentration range from 0.2 to 10 mu M (R = 0.9984) and 10-100 mu M (R = 0.9957) with a detection limit of 0.03 mu M (s/n = 3). The anodic peak current of UA was also a linear relationship in the concentration range 0.5-100 mu M (R = 0.9971) with a detection limit of 0.07 mu M. Moreover, the modified electrode surface has very good reproducibility and stability. (C) 2011 Elsevier B.V. All rights reserved.