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Rice straw can be utilized to use as a raw material for production of biofuel due to its content with a high amount of sugar in a form of 35-40% w/w cellulose and 25-30% w/w hemicelluloses. The effect of ultrasonic treatment to enhance the saccharification process of the enzymatic hydrolysis from rice straw was investigated. Two frequencies at 20 and 24 kHz of ultrasonic treatment were used for 80 min on rice straw before the enzymatic hydrolysis was applied by using commercially available enzyme cocktails: Accellerase™ 1000, Cellic CTec and NS 50013 at 50°C for 48 h. The result was found that the glucose concentration of pretreated rice straw was significantly higher than that of untreated rice straw (p <; 0.0001). At frequency of 24 kHz, the glucose concentration of all enzymes was significantly greater than that of enzymes at frequency of 20 kHz (p<;0.0001). When treating the rice straw with the Accellerase™ 1000 was yield significantly the highest glucose concentration among the other enzymes (p<;0.0001). These results reviewed that the ultrasonic pretreatment could enhance the saccharification process resulting in the acceleration of enzymatic hydrolysis.