With the requirements of green chemistry, more researchers are committed to studying green eutectic solvents (DES). Choline chloride eutectic solvent (DES) has been used in many fields because of its non-toxic, pollution-free and easy preparation. DES formed by choline chloride (ChCl) and glycerol (Gly) was studied in this paper. Comparing the infrared spectra of ChCl, Gly and ChCl/Gly, it was found that NC4 and O—H had red-shift and the combination and disappearance of characteristic peaks, which all indicated that ChCl/Gly formed DES through N—H+Ch…Cl- and Cl-…H—OGly. In order to explore the changes of DES at different temperatures, the infrared spectral data of ChCl/Gly at 25～135 ℃ were tested with temperature as an external disturbance. It was found that the intensity, width and position of NC4 and O—H spectral peaks changed to a certain extent at different temperatures, but these characteristic peaks were all wide peaks with overlapping phenomenon. It is difficult to conduct one-dimensional spectral analysis, but two-dimensional infrared spectroscopy (2D-IR) can better analyze the complex solution system and explore its formation mechanism and intermolecular structure. 2D-IR analysis was performed on the data of NC4 and O—H characteristic peaks in ChCl/Gly as a function of temperature to obtain the dynamic change sequence and predict the different molecular clusters in the solution. The results Three automatic O—H peaks at 3 539, 3 380 and 3 177 cm-1 at 25~65 ℃. Different automatic peaks of NC4 also appear, which can be attributed to ChCl/Gly, ChCl/(Gly)2 and ChCl/(Gly)3. At 65~105 ℃, O—H has two automatic peaks at 3 539, 3 380 and 3 400 cm-1, and NC4 has corresponding changes, which can be attributed to ChCl/Gly and ChCl/(Gly)2. At 105~135 ℃, O—H only had a strong automatic peak at 3 380 cm-1, and NC4 only had an automatic peak, attributed to ChCl/Gly. Through 2D-IR analysis, it is concluded that molecular clusters in the solution dissociate into a more stable structure with the rise of temperature. In order to verify this conclusion and explore the specific connection mode of N—H+Ch…Cl- and Cl-…H—OGly formed by Cl- between molecules of different clusters as Bridges. The density functional theory (DFT) was used to optimize the configuration geometry at the B3LYP/6-311G++(2d,p) theoretical level, and the relevant thermodynamic data were calculated, which confirmed the possibility of the existence of different molecular clusters in ChCl/Gly. The study shows that the combination of 2D-IR and density functional theory calculation can be well used to analyze the law of different molecular clusters in DES with temperature change, which better solves the problem that it is difficult to analyze overlapping and wide peaks in a one-dimensional infrared spectrum under temperature disturbance.