Gold nanoparticles (GNs) could be efficiently immobilized on binary mixed self-assembled monolayers (SAMs) on a gold surface composed of 1,6-hexanedithiol and 1-octanethiol (nano-Au/SAMs gold electrode). This GN chemically modified electrode was used for electrochemical determination of ascorbic acid (AA) and dopamine (DA) in aqueous media. The result showed that the GN-modified electrode could clearly resolve the oxidation peaks of AA and DA, with a peak-to-peak separation (∆Ep) of 110 mV enabling determination of AA and DA in the presence of each other. The linear analytical curves were obtained in the ranges of 0.3–1.4 mM for AA and 0.2–1.2 mM for DA concentrations using differential pulse voltammetry. The detection limits (3σ) were 9.0×10-5 M for AA and 9.0×10-5 M for DA.