A novel Au-nanoparticles/poly-(2-amino-2-hydroxymethyl-propane-1,3-diol) film modified glassy carbon electrode (AuNPs/poly(trisamine)/GCE) was constructed for the simultaneous determination of norepinephrine (NE), acetaminophen (AC) and L-tyrosine (Tyr) by differential pulse voltammetry. Electrochemical impedance spectroscopy and scanning electron microscopy indicate that the trisamine film was successfully polymerized on the surface of GCE and the film efficiently decreased the charge transfer resistance value of electrode and improved the electron transfer kinetic between analytes and electrode. The separation of the oxidation peak potentials for NE-AC and AC-Tyr were about 160 mV and 240 mV, respectively. The calibration curves for NE, AC and Tyr were obtained in the range of 1.3-230.1 mu mol L-1, 1.90-188.0 mu mol L-1, and 3.9-61.8 mu mol L-1, respectively. The detection limits (S/N = 3) were 0.0711 mu mol L-1, 0.1 mu mol L-1 and 0.9 mu mol L-1, for NE, AC and Tyr, respectively, The diffusion coefficient and the catalytic rate constant for the oxidation reaction of NE at AuNPs/poly(trisamine)/GCE were calculated as 1.55 (+/- 0.2) x 10(-6) cm(2) s(-1) and 2.28 (+/- 0.17) x 10(3) mol(-1) Ls(-1), respectively. Finally, AuNPs/poly(trisamine)/GCE was satisfactorily used for the determination of NE, AC, and Tyr in pharmaceutical and biological samples. (C) 2014 Elsevier B.V. All rights reserved.