The process of ATP biosynthesis from adenosine catalyzed by Saccharomyces cerevisiae was studied using an efficient energy regeneration system. A fractional factorial design (2^(9-5)) was used to evaluate the effects of different components in the medium. Magnesium chloride, toluene, and acetaldehyde were found to significantly influence ATP production. The concentrations of the three factors were then optimized using central composition design and response surface analysis. Based on the second-order polynomial model obtained from the experiments, the optimal parameters were obtained as follows: adenosine 20 g/L; glucose 67 g/L; S. cerevisiae cells 250 g/L; magnesium chloride 4.37 g/L; potassium dihydrogen phosphate 67 g/L; toluene 1.40 mL/L; acetaldehyde 2.67 mL/L; pH 7.0; and temperature 37.0 ℃. Under the condition, the yield and concentration of ATP reached 97.5% and 37 g/L, respectively. The yield was nearly 10% higher than the level before optimization and the concentration increased two-fold. In addition, the utilization efficiency of energy after optimization increased nearly 6%.