The electrochemical oxidation of dopamine (DA) and serotonin (ST) have been investigated by application of Nafion/Ni(OH)2-multiwalled carbon nanotubes modified glassy carbon electrode (Nafion/Ni(OH)2-MWNTs/GCE) using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronoamperometry (CA) methods. The modified electrode worked as an efficient electron-mediator for DA and ST in the presence of ascorbic acid (AA). Voltammetric techniques separated the anodic peaks of DA and ST, and the interference from AA was effectively excluded from DA and ST determination. The DPV data showed that the obtained anodic peak currents were linearly proportional to concentration in the range of 0.05–25 μmol L−1 with a detection limit (S/N = 3.0) of 0.015 μmol L−1 for DA and in the range of 0.008–10 μmol L−1 and with a detection limit of 0.003 μmol L−1 for ST. The proposed sensor was used for determination of ST and DA in human blood serum with satisfactory results.