Fresh corn stover, which could be separated into fermentable juice and lignocellulosic bagasse, is considered as an alternative raw material for second-generation ethanol production. However, the influence of harvest time of corn stover on ethanol yield has not been evaluated yet. In this work, aiming to improve ethanol yield from fresh corn stover, the harvest time was redefined and optimized. Liquefaction plus simultaneous saccharification and co-fermentation (L + SScF) was carried out to define the realistic ethanol yields. Results showed that compared with the corn stover harvested in the end of physiological stage, the conventional harvest stage, the corn stover harvested in dent stage provided the highest theoretical ethanol yield of 9.92 t/ha (5.64 t/ha for the case that exclude xylan derives). 3.53 t/ha of ethanol was produced from the fresh stover harvested in dent stage, which was 1.64 times higher than using the stover obtained in the conventional harvest time. This study demonstrated that bio-ethanol yield was hugely influenced by harvest time of corn stover, and the stover harvested in dent stage has the highest ethanol yield. (C) 2019 Elsevier Ltd. All rights reserved.