Use of starch biomass in fuel ethanol production is very promising due to its economic viability and availability. Starch hydrolysis step is required before the fermentation process because the yeast Saccharomyces cerevisiae is not able to convert directly starch into ethanol. This study has analyzed the performance of alpha-amylase enzyme in the step of starch liquefying, varying the reaction time. The samples were analyzed by high-performance liquid chromatography (HPLC). The highest glucose concentration (130,16 g/L) was obtained after 1 hour of hydrolysis enzymatic at 90 ˚C, pH 5.5, using Termamyl 2X followed by total hydrolysis with AMG 300 at 60 ˚C and pH 4.5.