Based on one parameter at a time, saccharification of delignified sorghum biomass by 4% and 70% v/v sulfuric acid resulted in maximum 30.8 and 33.8g% sugar production from biomass respectively. The Box Behnken Design was applied for further optimization of acid hydrolysis. As a result of the designed experiment 36.3g% sugar production was achieved when 3% v/v H2SO4 treatment given for 60min at 180°C. The process was scaled-up to treat 2kg of biomass. During the screening of yeast cultures, isolate C, MK-I and N were found to be potent ethanol producers from sorghum hydrolyzate. Culture MK-I was the best so used for scale up of ethanol production up to 25L capacity, which gave a yield of 0.49g ethanol/g sugar from hydrolyzate obtained from 2kg of sorghum biomass.