The medium for the production of 2-phenylethanol (2-PE) by Kluyveromyces marxianus strain LY1 were studied with the single factor and response surface methodology. The optimized medium contains 8.39?g/L L-phenylalanine, 43.25?g/L saccharose, 6.68?g/L yeast extract. The yield of 2-PE reached up to 1.450?g/L, increased by 51.0% compared with original condition. Then strain LY1 was further analyzed for its tolerance properties and high temperatural fermentation ability. The results showed that the highest tolerance to 2-PE of strain LY1 was 2.0?g/L at 36?℃ and 1.5?g/L at 42?℃. Moreover, the yield of 2-PE reached to 0.809?g/L at 42?℃. It would provide reference for production of 2-PE by thermotolerant yeasts at high temperature.