Bio-ethanol was produced at a broad temperature range of 30–60◦C but was highly specific to a pH range of 6.0–7.0. The final bio-ethanol production at 30, 40, 50, and 60◦C was 3.37, 3.83, 1.94, and 1.60 g/L, respectively, when a 1% glucose solution was used, and the yield increased significantly with increasing cell-free enzyme concentrations. A comparative study revealed better results for the conventional fermentation system (4.46 g/L) at 30◦C than the cell-free system (3.37 g/L); however,the efficacy of the cell-free system increased with temperature, reaching a maximum (3.83 g/L) at 40◦C, at which the conventional system could only produce 0.48 g/L bio-ethanol. Successful bio-ethanol production using a single yeast cell-based enzyme system at higher temperatures will lead to the development of novel strategies for efficient bio-ethanol production through SSF.