Abstract: As a renewable and clean energy, bio-ethanol has been received widespread attention in recent years. Ethanol fermentation at high temperature can reduce the consumption of cooling water and energy in the process of fermentation, it also solve the problems caused by saccharification and uncontrolled fermentation temperature, resulting in the simultaneous saccharification and fermentation of cellulosic ethanol. Therefore, the fermentation period can be shorten and the production cost be reduced. A thermotolerant yeast strain HN-1 isolated from rotting tobacco leaves was identified and its ethanol fermentation characteristics was investigated in this study. The phylogenetic analysis indicated that phylogenetic phylogenetistrain HN-1 behaved like Issatchenkia orientalis. This strain useed glucose and D-fructose but could not use xylose, maltose, lactose, sucrose, starch and cellobiose as sole carbon source to produce ethanol. When HN-1 was cultivated in 50 g/L glucose liquid medium, the highest biomass was obtained at 35 and 38℃. Increasing the cultivation temperature to 42 and 45℃ decreased the biomass growth and intensified the cell death. There was no major difference between ethanol productions (21.43~23.12 g/L) fermented at 35~45℃ from 50 g/L glucose. When increasing the fermentation temperature, the ethanol productivity was increased from 0.31 g/(L·h) at 35℃ to 0.65 g/(L·h) at 45℃ after 24h fermentation. The fermentation time was shortened. When the fermentation was conducted at 38℃, 28.77, 43.21, 58.19 and 59.53 g/L ethanol were produced from 60, 90, 120 and 150 g/L glucose with yields of 0.457, 0.468, 0.460 and 0.386 g/g and productivities of 0.38, 0.39, 0.48 and 0.40 g/(L·h) The results also indicated that fed batch fermentation could not enhance the ethanol production. Strain HN-1 could utilize corn straw hydrolysate with 43.08 g/L glucose and 27.13 g/L xylose to produce 20.74 g/L ethanol. The glucose conversion rate was 0.468 g/g, which was 91.6% of the theoretical yield. Noxylose was utilized during the fermentation. This research provides a valuable thermotolerant strain. It is expected that it will be beneficial for industrialized production of bio-ethanol with high temperature fermentation.