AbstractAcorn starch was used for ethanol production by separate hydrolysis and fermentation (SHF) in this study. The influence of tannins on hydrolysis and fermentation was investigated using ultrasonic-assisted extraction (UAE) to decrease the amount of tannin before SHF. The tannin was shown to have a negative role in hydrolysis and fermentation, and UAE can improve the two processes. The tannin content of acorn starch decreased from 6.19% to 1.91% with the UAE pretreatment time of 200 min. When the pretreatment time was 120 min, the glucose concentration increased from 78.08 to 98.76 g/L after 24 h of hydrolysis. The highest ethanol concentration was 42.22 g/L, which was obtained from the same pretreated acorn flour fermented for 12 h. However, the maximum ethanol yield was 88.06% of the theoretical yield, while pretreatment time was 80 min. Scanning electron microscope images indicated that protein was separated from the starch granules by UAE, as well as by the molecular weight of starch which decreased significantly based on the results from gel permeation chromatography (GPC) analysis.