Abstract: In order to promote the development and utilization of distiller’s grain resources in China, the present study explored the enzymatic hydrolysis conditions of distiller’s grains cellulose based on ultrasonic pretreatment. Firstly, the effects of ultrasound parameters (time, temperature, power) and enzymatic hydrolysis process parameters (time, pH, temperature, enzyme addition, substrate concentration) on the enzymatic hydrolysis of vinasse cellulose were studied through single-factor experiments. The Plackett-Burman test was carried out to screen out the key factors which affected the enzymatic hydrolysis reaction, and then the Box-Behnken test was used to optimize the enzymatic hydrolysis process. The Plackett-Burman test results showed that the key factors affecting the enzymatic hydrolysis of vinasse cellulose were ultrasound time, enzymatic hydrolysis time, enzymatic hydrolysis pH, and enzyme addition. The Box-Behnken results showed that the optimal enzymolysis process conditions were ultrasonic time 21 min, enzymolysis time 140 min, enzymatic hydrolysis pH5.41, enzyme addition 990 U/g, ultrasonic temperature 60 ℃, ultrasonic power 200 W, enzymolysis temperature 50 ℃, and the concentration of enzymatic hydrolysis substrate 1:20 g/mL. Under these conditions, the cellulose conversion rate of distiller’s grains were 5.62%, which was 35.4% higher than that of the control group. The results showed that ultrasonic pretreatment was an effective way to improve the cellulose conversion rate of distiller’s grains.