Raman spectroscopy is used to characterize the process of ethanol fermentation and look into the Saccharomyces cerevisiae cells at single-cell level. The ethanol fermentation using high-concentration cassava starch as feedstock is processed in a 500 L fermentor and the Raman spectra of substrate, product and individual yeast cell are acquired by using laser tweezers Raman spectroscopy (LTRS). Major results are as follows: 1) Raman spectroscopy can monitor the dynamic changes of substrate and product during the ethanol fermentation; 2) the changes of intracellular components of yeast cells exhibit three stages, i.e., primary fermentation, main fermentation and later fermentation, which are similar to those of products, but there is a delay of about 4 h; 3) yeast cells amend its physiological state and intracellular compounds to adapt to the high osmotic stress at the initial stage and the high ethanol concentration at the later stage of fermentation. Random coiling dominates the secondary structure of yeast protein as the ethanol concentration increases; 4) purine is synthesized and accumulated in yeast cells at the later stage, but the amounts of individual cells are greatly heterogeneous. This work provides beneficial reference on the ethanol fermentation from a new perspective. LTRS shows the potential to open a new frontier of microbial fermentation.