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The alternative sweetener used to beverage and food with a calorie controlled, to control weight loss program or to prevent tooth decay. The alternative processes that are currently being developed are producing of xylitol from xylan of the cellulose biomass. The goal of this study is to determine the most influential process variable and optimum operating condition of xylitol production through enzymatic hydrolysis reaction of corn cob waste. The parameters are the time (30-120 minutes substrate ratio:starter (1:5-1:15 w/w). The result showed a high number of cell on a substrate concentration of 10% led to increase in xylose consumption while the xylitol production is not too large because the substrate is used for yeast cell growth so that production of xylitol to be reduced and the efficiency is low. The high efficiency (25%) occurred in 20% of substrate concentration because the xylose consumption is used for growth and xylitol production. While the concentration of 30% decline in the consumption of xylose by yeast compared with others and the efficiency is not too high. This is because the high concentration of xylose which led to substances inhibitor in the media and the production of xylitol become low.