Molecular level studies have the potential to reveal the protective mechanism of cryoprotectants during the cryopreservation of bio-samples. The Raman spectra of the VS55 solution with different concentrations (8.4 M ~ 0 M) are captured as the temperature decreasing. The distinguished ice crystal peaks are recognized for the low-concentration solutions with the temperature decreasing, and the high-density amorphous structure peak of water molecules is found for the high concentration solution. And the marker peaks of the different solute substances are distinguished by comparing the pure component spectra with the mixed aqueous solution. The S O bond in DMSO molecules is easy to form the DMSO-Water hydrogen bond structure and the C N bond indicates that the formamide aggregate structure decomposes as the temperature decreases. Moreover, the abnormal shift phenomenon of S O and C N bond during the crystallization is probably due to the transformation of the dominant hydrogen bond structure in solution systems and the concentration effect of residual solution. The molecular interaction of different components in cryoprotectant mixture will provide an important supplement to understand the dynamic behavior of cryoprotectant and further explore the mechanism of the cryopreservation biological samples.