The acorn starch physicochemical properties were studied, and the acorn flour was used to ferment fuel ethanol by tannin tolerance mutants. The results showed that the gelatinization temperature of acorn starch lower than cassava starch and corn starch, which indicated that the gelatinization of acorn starch is easier than corn starch and cassava starch; Two tannin-tolerance mutant strains P05 and P18 were obtained after UV irradiation and tannin gradient domestication. Use of mutant P18 may decrease the extraction times of tannin from 5 to 3 times, which simplifies the production process and reduces the ethanol production cost.