Background: The yeast Saccharomyces cerevisiae is a promising host cell to produce 2,3-butanediol (2,3-BDO). However, the fermentation environment restricts 2,3‑BDO yield, productivity, and titre from engineered yeast. In the present study, we propose a strategy in which a suitable dissolved oxygen content and acid stress level can improve the 23-BDO yield of S. cerevisiae W141. Five different concentrations of short-chain fatty acids were evaluated and noxE overexpression was performed to disrupt the intracellular redox balance and alter the NADH content associated with 2,3‑BDO synthesis, which can significantly increase or inhibit 2,3‑BDO yield.Results: The five assayed short-chain fatty acids have different effects on the fermentation characteristics of yeast, were formic, butyric and valeric acids can inhibit the synthesis of 2,3‑BDO. Only low concentrations of acetic and propionic acids could significantly increase the yield of 2,3‑BDO, especially when 1 g/L acetic acid was added, which stimulated the expression of acid stress-related genes in S. cerevisiae W141 (haa1p and hog1p) and increase the 2,3-BDO yield by 29.74%. To further verify that acid stress primarily disrupts the intracellular redox balance by altering the NADH content, we constructed a S. cerevisiae strain, W141-E, which overexpresses the noxE gene of Lactobacillus. After adding 1 g/L acetic acid, the 2,3‑BDO yield from in S. cerevisiae W141-E increased by 43.64%, confirming the validity of our strategy. When the optimized fermentation oxygen content was 0.6 vvm, the 2,3‑BDO yield from S. cerevisiae was greatly improved after the addition of acetic acide.Conclusions: In the present study, we demonstrated that a suitable dissolved oxygen and acid stress are highly effective for increasing the 2,3-BDO yield from S. cerevisiae W141. 2,3-BDO biosynthesis was heavily dependent on the intracellular NADH content, which is closely associated with glycolysis and the TCA cycle and is likely important for the production of 2,3-BDO by S. cerevisiae.