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We report here preparation and characterization of an AuCu3 alloy electrochemical sensing platform fabricated on an easy for mass production barrel-plated gold electrode (AuBPE). Note that the AuBPE consists of Cu main body with successive plating of a very thin layer of Ni and Au (i.e., Cu–Ni–Au multilayer). By suitably manipulating the grain boundary diffusion between individual metallic layers of Cu–Ni–Au by means of thermal annealing treatment, AuCu3 alloy film can be formed directly on the outer surface of the AuBPE. Electrochemical activation in alkaline solution can further generate a nanostructured AuCu3 alloy film. This facile and novel approach is promising to prepare sensors for electroanalytical applications, e.g., glucose oxidation in pH-neutral solutions and simultaneous detection of uric acid, dopamine and ascorbic acid.