Four different Au/Fe-Zn-Mg-Al-O hydrotalcites (HTs) supported Au catalysts were synthesized by the incipient impregnation method and characterized by X-ray photoelectron spectroscopy and scanning electron microcopy. Enhancement of biohydrogen production was found by addition of HTs supported Au catalyst within a concentration range of 83-833mg/L, depending upon the composition of HTs. An active and stable HTs supported Au catalyst was achieved in the presence of Zn on uncalcined support. The Au/Zn-Mg-Al HT exhibited a maximum hydrogen yield of 2.74±0.14mol H2/mol sucrose at a dose of 167mg/L, which was two times higher than the control. This behavior probably is related to the combination effects of the metallic dispersion of Au on the support surface containing Zn, which can act as the active sites of hydrogenase enzyme. Calcined Zn-Mg-Al HT did not promote biohydrogen production with Au catalyst loading.