Deoxyadenosine triphosphate (dATP) is an important biochemical molecule. In this paper, the synthesis of dATP from deoxyadenosine monophosphate (dAMP), catalyzed bySaccharomyces cerevisiae,was studied. By adding chemical effectors, an efficient ATP regeneration and coupling system was constructed to achieve efficient synthesis of dATP. Factorial and response surface designs were used to optimize process conditions. Optimal reaction conditions were as follows: dAMP 1.40 g/L, glucose 40.97 g/L, MgCl2·6H2O 4.00 g/L, KCl 2.00 g/L, NaH2PO431.20 g/L, yeast 300.00 g/L, ammonium chloride 0.67 g/L, acetaldehyde 11.64 mL/L, pH 7.0, temperature 29.6 °C. Under these conditions, the substrate conversion was 93.80% and the concentration of dATP in the reaction system was 2.10 g/L, which was 63.10% higher than before optimization, and the concentration of product was 4 times higher than before optimization. The effects of glucose, acetaldehyde, and temperature on the accumulation of dATP were analyzed.