In this study, Au-nanoparticles/poly-Trypan Blue modified glassy carbon electrode (AuNPs/poly-TrB/GCE) was fabricated for the simultaneous determination of cysteine (Cys), uric acid (UA) and tyrosine (Tyr). There are limitations in bare glassy carbon electrode (GCE) in showing clear oxidation peaks for Cys, UA and Tyr; whereas AuNPs/poly-TrB modified electrode completely separated the voltammetric signals of Cys, UA and Tyr at +0.40, +0.58 and +0.90 V, respectively (vs. Ag/AgCl electrode). This modified electrode shows excellent electrocatalytic activity towards the oxidation of Cys with a potential shift about 460 mV to a less positive potential. Under optimized experimental conditions, the detection limits of Cys, UA and Tyr were calculated as 0.006, 0.07 and 0.008 mu mol L-1, respectively, at this simple construction sensor. Finally, the fabricated sensor was satisfactorily used for the simultaneous determination of these molecules in human biological samples. (C) 2017 Elsevier B.V. All rights reserved.