Glucose syrup is a valuable food ingredient produced by the hydrolysis of starch preferrably from maize. In this study, small-scale production process of glucose syrup from wheat, maize and potato starches was investigated. Two-step ezymatic hydrolysis using α-amylase and amyloglucosidase for liquefaction and saccharification, respectively, was analyzed based on the glucose content of a final product. The optimization of conditions was conducted with different initial amount of starch, different amount of enzymes and reaction time. Starch slurries at 30% were hydrolzed into smaller dextrins by 0.0002% (mL/g, venzyme /wstarch) α-amylase for 2 hours and further hydrolyzed into glucose by 0.0002% (mL/g, venzyme /wstarch) amyloglucosidase for 48 hours optimally. These process conditions yielded glucose syrups with dextrose equivalent (DE) values of 97.04, 97.27 and 95.34% and dry matter content of 84.30, 78.30 and 82.37% from wheat, maize and potato starches, respectively. It was concluded that starch from different biological origins offered promising raw materials for the enzymatic production of glucose syrup wih high DE value at optimum conditions.