A highly selective and sensitive electrochemical sensor, AgNPs–penicillamine–Au, was developed for the simultaneous detection of dopamine (DA) and epinephrine (EP) in the presence of a high concentration of ascorbic acid (AA) and uric acid (UA). Microscopy and voltammetry techniques were used for the characterization of the modified electrode. Chronoamperometry was used for the determination of DA and EP in the linear range of 0.1 to 100.0 μM with detection limits of 0.2 nM and 0.5 nM, respectively. The simultaneous determination of DA, EP, AA and UA was achieved by using differential pulse voltammetry. The proposed sensor was successfully applied for the simultaneous determination of DA and EP in human blood sample with excellent recovery.