To comprehend the resistance mechanism of ethanol of Saccharomyces cerevisiae, Raman spectroscopy and singlecell analysis technique were used to record the Raman spectra of individual yeast cells during ethanol fermentation under different hyperosmosis caused by nonfermentable substrate,monitor the dynamic of major intracellular biomacromolecules. Increasing osmotic pressure significantly delayed the growth of yeast cells, substrate consumption and product formation, but the final ethanol production was not lower than control group even supplemented with 2.0 mol/L of sorbitol. Principal component analysis revealed that hyperosmosis mainly impact the peaks 1 300~1 306, 1 443 cm－1 and other Raman peaks derived from lipids, indicating the hyperosmosis may affect the synthesis of intracellular lipids of yeast. The intensity dynamic of Raman peaks showed that hyperosmosis impacted the synthesis period of macromolecules and the strength of peaks 782,1 301,1 602 and 1 657 cm－1 and then affected the metabolism direction of yeast cell. The results indicate that high permeabilityresistant yeast strains can adapt to hypertonic environment, adjust the content of intracellular components to achieve high yield fermentation.