Methanobactin (Mb) is a copper-binding peptide that appears to function as an agent for copper sequestration and uptake in methanotrophs. Mb can also bind and reduce Au(III) to Au(0). In this paper, Au/Al2O3 catalysts prepared by a novel incipient wetness-Mb-mediated bioreduction method were used for glucose oxidation. The catalysts were characterized, and the analysis revealed that very small gold nanoparticles with a particle size <4 nm were prepared by the incipient wetness-Mb-mediated bioreduction method, even at 1.0% Au loading (w/w). The influence of Au loading, calcination temperature and calcination time on the specific activity of Au/Al2O3 catalysts was systematically investigated. Experimental results showed that decomposing the Mb molecules properly by calcinations can enhance the specific activity of Au/Al2O3 catalysts, though they acted as reductant and protective agents during the catalyst preparation. Au/Al2O3 catalysts synthesized by the method exhibited optimum specific activity under operational synthesis conditions of Au loading of 1.0 wt % and calcined at 450 degrees C for 2 h. The catalysts were reused eight times, without a significant decrease in specific activity. To our knowledge, this is the first attempt at the preparation of Au/Al2O3 catalysts by Mb-mediated in situ synthesis of gold nanoparticles.