A novel phosphate amperometric nanobiosensor, based on an intimate integration of pyruvate oxidase (PyOx) and its cofactors, thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), with a highly ordered gold nanowires array (AuNWA) has been developed. The successful integration of PyOx and the co-factors, via crosslinking with bovine serum albumin (BSA) and glutaraldehyde (GLA), onto the AuNWA was confirmed by cyclic voltammetry and amperometry. The resulting nanobiosensor achieved a detection limit of 0.1μM, a linear concentration range of 12.5-1000μM, and a sensitivity of 140.3μAmM-1cm-2. Notably, the incorporation of the AuNWA reduced the required PyOx concentration by 70-120 fold and the presence of common interferants, such as chloride, sulfate, fluoride, nitrite and nitrate ions did not interfere with phosphate detection. Furthermore, the nanobiosensor demonstrated a very high stability with repeated use over two weeks and was successfully used for the determination of phosphate in water samples with an average recovery of 96.6±4.9%.