In this article, a novel electron transfer mediator based on the ferrocene-functionalised graphene oxide (Fc-GO) was successfully synthesised by the amide linkage between amino (–NH2) in m-ferrocenylaniline (FcAni) and carboxylic (–COOH) in GO sheet through coupling reagents. This novel electroactive Fc-GO can effectively prevent the ferrocene mediator leaking from the electrode surface. Then, gold nanoparticles assembled ferrocene-functionalised graphene oxide (AuNPs@Fc-GO) was prepared and further confirmed by transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDS), Fourier transform-infrared spectroscopy (FTIR) and X-ray powder diffractometer (XRD). The AuNPs@Fc-GO was modified on the surface of glassy carbon electrode (GCE) to construct an electrochemical sensor for sensitively simultaneous determination of dopamine (DA) and acetaminophen (AC). The characteristics toward DA and AC were investigated by the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. Simultaneous determination of both analytes was accomplished under the optimum condition. The oxidation peak potential of DA and AC was 0.20 and 0.43 V, respectively. The linear current response was obtained in the concentration range of 2–200 μM with the detection limit (S/N = 3) of 0.32 μM for DA and 2–200 μM with the detection limit of 0.21 μM for AC, respectively. Finally, the AuNPs@Fc-GO modified GCE was applied for the determination of DA and AC in human urine samples with satisfactory results. Therefore, the AuNPs@Fc-GO was a novel promising candidate for fabricating an electrochemical sensor for the determination of DA and AC in real biological samples.