This study was conducted at the laboratories of the Faculty of Agriculture, Damascus University and the National Commission of Biotechnology in 2012 to investigate he possibility of Production of bioethanol using local isolate of Saccharomyces cerevisiae from raw sugar refining molasses and to determine the effect of different temperatures, pH and concentration of Dry matter (Brix degree) in molasses on the amount of ethanol produced. A local isolate (R1) of Saccharomyces cerevisiae selected previously due to its superiority in ethanol production was taken from wine brewing residues and exposed to different temperatures (20, 25, 30 and 35OC), pH (4, 4.5, 5 and 5.5) and the Brix degrees (16, 20, 24 and 28%) during fermentation. Results revealed that the best amount of ethanol was produced at temperature of 30 º C, pH 5, and Brix degree in molasses 16%.