Abstract: This paper studied the effect of ethanol stress on the growth situation of Saccharomyces cerevisiae, the Raman spectroscopic of its cells were got and analyzed by laser tweezers Raman spectroscopy (LTRS) , and the proteomic changes in its cells were analyzed at the molecular level. The results showed that the ethanol could inhibit yeast growth. With the ethanol concentration increasing, the yeast cell diameter decreased, stable period delay, biomass and protein content also showed a decreasing trend. The relative content of alcohol concentration and biochemical composition in Saccharomyces cerevisiae cells could be obtained used LTRS as tool. 22 differential bands were detected in different concentrations of ethanol by SDS-PAGE, of which7 differential bands were identified by mass spectrometry. The 7 proteins function mainly related to telomere stability, cell autolysis and key metabolic. Different concentrations of ethanol could induce the same protein expression changes, such as HSP104 protein, which indicated the metabolic pathway of these proteins participate play a universal role in yeast ethanol tolerance.