The exact determination of lactate concentration is very important in the fields of food quality and clinical diagnosis. A non-enzymatic amperometric sensor based on nanostructured porous Cu-Au electrocatalyst martial was designed and employed for lactate determination. For this purpose, the bimetallic surface was successfully coated on the glassy carbon electrode (GCE) using co-electrodeposition of copper and gold ions. The Cu-Au alloy proved to be an effective interface for the direct electrochemical oxidation of lactate. The Cu-Au modified GCE exhibits excellent lactate sensing capabilities thanks to the excellent conductivity of gold element in bimetallic material and high surface area of the porous Cu-Au alloy. In phosphate buffer solution, this novel electrochemical lactate sensor demonstrates a linear response to lactate within the concentration range of 20 to 2000 µM. The detection limit (based on S/N = 3) of the assay was estimated to be 5 µM. The established electrochemical sensing protocol is a highly selective device for the analysis of lactate in biological fluids. The lactate level in saliva samples was successfully quantified before and after exercise of athletes using the recommended strategy. The present non-enzymatic sensor offers a convenient, fast, cost-effective, and effective protocol for lactate measuring in clinical diagnosis applications.