Well aligned Pt/Au alloy nanowire arrays are successfully synthesized and used as the substrate for the construction of phosphate biosensors. Pyruvate oxidase is immobilized onto the nanowire surface together with co-factors via cross linking approach. Morphology, composition, structure and chemical state of the nanowires are characterized by SEM, TEM, EDS and XPS, and the electrochemical behaviors of the as-prepared phosphate biosensors are investigated by cyclic voltammogramic and chronoamperometric techniques. Especially, the influence of the composition and aspect ratio of Pt/Au alloy nanowire arrays on the response of phosphate biosensors are studied as well. The present phosphate biosensor shows a high sensitivity of 50.5 μA· cm-2 ·mM-1 and a wide linear range between 248 and 1456 μM toward the detection of phosphate. In addition, the biosensor also exhibits good selectivity and stability, which promises great potential in phosphate detection applications.