The selective oxidation of D-galactose to galactonic acid over Au/Al2O3 was studied isothermally in a semi-batch shaker reactor under pH-controlled conditions at atmospheric pressure. A series of Au/Al2O3 catalysts were prepared and calcined at different temperatures to achieve different gold-particle sizes. The catalytic properties of the gold nanoparticles were affected by the cluster size. A detailed comparison of activity and selectivity of these catalysts was made, which demonstrated that Au/Al2O3 with a mean particle size of 2.6 nm exhibited the highest activity. The influence of the pH value of the reaction medium on the selective oxidation of D-galactose was elucidated. Alkaline conditions were characterized by high catalyst activity and selectivity to aldonic acid. Inhibition of the catalytic activity was observed in the acidic medium. The intermediate species was present at low pH values, and this resulted in low conversion and selectivity to the main product, galactonic acid. The electrochemical potential of the catalyst was correlated to the catalytic activity.