In this study, poly-Trypan Blue modified glassy carbon electrode was developed for the simultaneous determination of ascorbic acid (AA), noradrenaline (NA), acetaminophen (AC) and tryptophan (Try). Bare glassy carbon electrode (GCE) has limitations for resolving the oxidation currents of these compounds. Poly-Trypan Blue modified electrode not only separates the voltammetric signals of AA, NA, AC and Try with potential differences of 150, 170, and 280 mV between AA-NA, NA-AC, and AC-Try, but it can also enhance the oxidation currents of AA, NA, AC and Try by 11.0, 7.0, 1.4, and 3.8 times, respectively, compared to bare electrode. The diffusion coefficient, D, and the heterogeneous rate constant, K-h, were estimated for the oxidation of noradrenaline at the modified surface. The detection limits of AA, NA, AC and Try were also calculated as 0.10, 0.06, 0.10 and 0.80 mu mol L-1, respectively. Finally, the fabricated sensor was satisfactorily used for the simultaneous determination of these molecules in pharmaceutical and human biological samples. (C) 2016 Elsevier B.V. All rights reserved.