The main factors, including temperature, agitation rate, pH, and particles stuffing rate, were chosen in ethanol fermentation from stalk juice of sweet sorghum using immobilized S. cerevisiae in shaking flasks in order to determine suitable conditions. The results showed that a temperature of 34C, agitation rate of 150-200 r min-1, pH of 4.5, and particles stuffing rate of 25% should be selected as the suitable condition. The results of verification experiments at the selected condition demonstrated that the ethanol yield, the CO2 weight loss rate, and fermentation time were 95.15%, 0.508 g h-1, and 6 h in the shaking flasks, respectively. Meanwhile, the ethanol yield and the fermentation time were 96.72% and 10 h in the 5 L bioreactor. As a result, it could be concluded that the determined condition was suitable and reasonable for ethanol fermentation from by immobilized S. cerevisiae.