The amount of ethanol production in fermentation process, depends on environmental factors. In order to study the influential factors in producing ethanol by strain of Hanseniaspora opuntiae, the amount of ethanol production was measured in YPD culture medium (1 % yeast extract, 2 % peptone, 2 % glucose, in distilled water) at different temperature and pH and also in the presence of specific volume of different carbon sources including glucose, sucrose, fructose syrup, glucose syrup, molasses and whey, and different nitrogen sources such as ammonium phosphate, ammonium sulfate, industrial urea, ammonium nitrate, peptone and amine-chloride. In order to optimize the conditions of ethanol production and to gain maximum production rate by yeast strain, test design method with Response Surface Method was used, aiming at achieving better results, significant decrease in the number of tests and optimizing production conditions. Using the Placket-Burman option of Minitab © software, effectual factors were screened with. To determine the amount of ethanol produced used HPLC technique. The results showed that among the measured factors, nitrogen source has the most effects in producing ethanol by studied strain and the minimum effect was related to ph. Also, among the carbon sources, the highest production occurred in the presence of sucrose and molasses, and among nitrogen supplies the best performance in producing ethanol occurred with peptone and ammonium nitrate.