A green bioreductive approach with methanobactin was adopted to fabricate bimetallic Au-Pd/Al2O3 catalysts for solvent-free oxidation of glucose to gluconic acid with H2O2 at atmospheric pressure. The catalyst was characterised by diffuse reflectance UV-vis spectroscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction techniques to understand synergistic interactions between Au and Pd. Effects of Au to Pd molar ratio on the catalytic activity of Au-Pd/Al2O3 were investigated. The Au-Pd/Al2O3 catalyst with Au/Pd molar ratio of 0.8:0.2 exhibited excellent catalytic performance. With the catalyst, the oxidation activities of glucose to gluconic acid 2856 mmol min-1 g-1 and selectivity 99.6% were attained at 323 K with H2O2. The results indicated the activity and selectivity was affected by the ratio of Au/Pd on the Al2O3. The formation of Au0.8Pd0.2/Al2O3 was favourable for the catalytic reaction.