This study presents dark fermentative hydrogen gas production from acid hydrolysed waste paper towel hydrolysate. Firstly, Box-Behnken statistical experiment design was used to investigate glucose production from waste paper towel by acid hydrolysis. S/L ratio, hydrolysis time and pH were chosen as independent variables while glucose concentration in the hydrolysate was the objective function. Highest glucose concentration of 32.17 g/L was obtained at S/L ratio of 100 g/L, pH 0 (gauge pH) and 180 min hydrolysis time. All variables were found to have significant effect on glucose formation. Glucose concentration increased by increasing the S/L ratio and hydrolysis time, but decreased by increasing the pH. Secondly, the hydrolysate obtained at most convenient hydrolysis conditions was subjected to lime putty treatment for the removal of 5-HMF and SO42−. Thirdly, H2 gas was produced from the detoxified hydrolysate by dark fermentation. Maximum H2 formation yield, rate and H2 percentage in the gas phase were 1.02 mol H2/mol glucoseconsumed, 130.22 mL H2/g biomass.h and 43%, respectively. The remaining 5-HMF after lime treatment was effectively removed in dark fermentation.