The present study investigated the production conditions of bioethanol fermented from the enzymatically hydrolyzed polysaccharides (PS) of Ulva lactuca. U. lactuca powder (5%, w/v) was sequentially treated with 0.4 N HCl at 121°C for 20 min, cellulase hydrolysis, α-amylase hydrolysis, and MA103/MAEF108 crude enzymes hydrolysis. The total sugar content, reducing sugar content, and NaCl content of the U. lactuca PS hydrolysate were 7.39 mg/ml, 8.40 mg/ml, and 5.0%, respectively. After U. lactuca PS hydrolysate was fermented with 10% S. cerevisiae BCRC21685 at room temperature for 72 h, the ethanol concentration, ethanol yield, and reducing sugar utilization of the fermentation solution reached 0.91%, 19.34 g/100 g Ulva powder, and 53.5%, respectively. When the U. lactuca PS hydrolysate was fed-batch fermented with 10% S. cerevisiae BCRC21685 at room temperature for 120 h, the ethanol concentration, ethanol yield, and reducing sugar utilization of the fermentation solution reached 1.02%, 21.76 g/100 g Ulva powder, and 58.74%, respectively. Fed-batch fermentation can further enhance the bioethanol production from Ulva lactuca fermentation with S. cerevisiae.