Gold nanoparticles (Au NPs) decorated carbon nanofibers (CNFs) have been prepared by an electrospinning approach and then carbonized. The prepared Au-CNFs were employed to modifying a screen printed electrode (SPE) for simultaneous determination of ascorbic acid (AA), dopamine (DA) and uric acid (UA). Au NPs are uniformly dispersed on carbon nanofibers were confirmed by the structure and morphological studies. The modified electrodes were tested in cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronoamperometry (CA) to characterize their electrochemical responses. Compared to bare SPE, the Au-CNFs/SPE had a better sensing response to AA, DA, and UA. The electrochemical oxidation signal of AA, DA and UA are well separated into three distinct peaks with peak potential separation of 280 mV, 159 mV and 439 mV between AA-DA, DA-UA and AA-UA respectively in CV studies and the corresponding peak potential separation in DPV studies are 290 mV, 166 mV and 456 mV. The Au-CNFs/SPE has a wide linear response of AA, DA and UA in DPV analysis over the range of 5–40 μM ( R2 = 0.9984), 2–16 μM ( R2 = 0.9962) and 2–16 μM ( R2 = 0.9983) with corresponding detection limits of 0.9 μM, 0.4 μM and 0.3 μM at S/N = 3, respectively. The developed modified SPE based sensor exhibits excellent reproducibility, stability, and repeatability. The excellent sensing response of Au-CNFs could reveal to a promising approach in electrochemical sensor.