Multi-walled carbon nanotubes (MWCNTs) and gold nanoparticles (Au NPs) were assembled on glassy carbon electrode (GCE) to form homogeneous and stable multilayer films using a layer-by-layer assembled method by electrostatic interaction. Electrochemical impedance spectroscopy (EIS), Cyclic voltammetry (CV), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), and Ultraviolet-visible absorption spectroscopy (UV) were used for characterization of multilayer films. Electrocatalytic activity of uric acid (UA) was studied. UA and ascorbic acid (AA) of electrochemical signals were well separated on the modified electrode in pH 7.0 phosphate buffered solution (PBS). The oxidation peak potential of AA and UA was appeared at −91 and 247 mV vs. SCE on differential pulse voltammograms (DPVs), respectively. Response of UA was linear in the range from 4.0 × 10 −6 mol/L to 3.0 × 10 −3 mol/L and linear regression equation was i pa (µA) = 1.027 + 0.037 C (µmol/L) with a correlation coefficient of 0.992, the limit of detection for UA was 2.0 × 10 −6 mol/L.