Catalytic conversion of biomass-derived glycerol (GLY) to high value-added products (e.g., glyceric acid, GLYA) has attracted considerable attention. Herein, three Au nano-catalysts supported on mixed metal oxide (MMO) (denoted as Au/Ni3Al-MMO, Au/Co3Al-MMO and Au/Mg3Al-MMO) were prepared via a two-step process: Au supported on layered double hydroxides precursors (Au/LDHs) was synthesized using a deposition-precipitation approach, followed by a further reduction treatment to obtain Au/MMO samples. The optimized Au/Ni3Al-MMO catalyst exhibits prominent catalytic behavior towards selective oxidation of GLY to produce GLYA (conversion: 89.9%; yield: 57.8%; TOF: 5914 h−1), which stands at the highest level compared with previously reported Au-based catalysts. XPS, in situ DRIFTS, and in situ Raman verify the formation of charge-mediated Auδ+−oxygen vacancy (Auδ+−Ov) originating from electron transfer from Au atom to oxygen vacancy at the interface, whose concentration is dependent on the strength of metal-support interaction. Researches on the structure-property relationship on the basis of in situ FT-IR and kinetic isotope effect (KIE) further verify that the charge-mediated interfacial sites (Auδ+−Ov) play a crucial role in determining catalytic behavior, in which Auδ+accelerates the bond cleavage of the rate-determining step (α-C–H) while Ov actives oxygen to generate superoxide anion (O2−). This work gives a systematic knowledge of structure-property correlation and reaction mechanism, which would pave the way for rational design and preparation of efficient heterogeneous catalysts towards the chemical transformation of biomass-based polyols.