The high-concentration batch grade acclimation of activated sludge for bio-hydrogen production was investigated. Continuous hydrogen production at yields of 2.20 mol H-2/mol sucrose was attained at a sucrose concentration of 110 g/L using the activated sludge acclimated by high-concentration batch grade acclimation. The maximum hydrogen production rate was 1,200 ml H-2/L h, and the sucrose metabolism rate of anaerobic fermentation was expedited by high-concentration batch grade acclimation. Steady ethanol-type fermentation and predominant flora were naturally attained through high-concentration batch grade acclimation at higher substrate concentrations. The results indicated that natural conversion of predominant flora plays a key role in developing ethanol-type fermentation.