The pH level is a prominent factor affects the ethanol fermentation. Optical tweezers Raman spectroscopy is used to analyze the process of ethanol fermentation at the initial pH levels of 3.0, 4.5, 6.5. Major results from this work are as follows: 1) The pH 4.5 level get the highest ethanol production and the pH 3.0 get the lowest one. 2) Raman intensities of bio-macromolecules of yeast cells at all pH levels exhibite dramatic changes in the first 15 hours. However, at the later stage of the fermentation, the pH 3.0 level displayes strong Raman intensities of intracellular lipids and proteins while pH 6.5 level shows weak. This indicates that a portion of substrate may be transformed into intracellular storage material by yeast cells at lower pH level. 3) Principal component analysis reveals that fermentation pH influences the physiological status of yeast cells from the beginning of fermentation, and bands 1440 cm-1 and 1600 cm-1 are the prominent contributors to the component loadings in different fermentation stages. This suggests the pH level of medium may affect the synthesis of lipid and the respiratory metabolism of yeast cells, influence the metabolism consequently. The results indicate Raman spectroscopy and single-cell analysis can uncover the physiological mechanism of the microbial cell during the fermentation process and provide new and reference information for the ethanol fermentation.