Recovery of sulfuric acid is very important after biomass converted to sugar by acid hydrolysis. In this work, the separation of sulfuric acid from sulfuric acid/glucose solution was studied by electrodiaysis. Three chamber method, which requires both anion membrane and cation membrane, is the most commonly used in the electrodialysis process, but two chamber method using only an anion membrane was the focus of this study. Sulfuric acid was perfectly separated from a mixture of 10~30 wt% glucose and 1~3 M sulfuric acid by electrodialysis using two chamber method. The separation rate of sulfuric acid lineary increased with higher current density when the affect of diffusion and convection of the membrane was small. Without electric energy, 45% of sulfuric acid was separated by diffusion and convection only.