Abstract: In recent years, the compound specific isotope analysis (CSIA) technology can be directly used to determine the stable isotope ratio of the element to be measured in a specific compound in the reaction system, and the analysis results are not affected by the reaction environment and have high accuracy. Therefore, this technology is not only applied to the transformation of environmental organic pollutants, but also gradually applied to the study of specific chemical reaction mechanism. In this paper, the carbon isotope ratio analysis method of specific target compounds in the liquefied products of glucose was established by C-CSIA, and the standard deviation of δ¹³C test value of the specific compounds was less than 0.3‰. The δ¹³C value and fractionation characteristics of intermediate products 1,4;3,6-dianhydro-alpha-d-glucopyranose (DGP) were compared with different liquefaction temperatures. At low temperature, the stable isotope ratio of the DGP has significant carbon isotope dilution effect, and the DGP gradually has carbon isotope enrichment effect when the temperature increases. At 140 ℃, the δ13C value of the DGP decreases from (-15.89±0.08)‰ to (-17.79±0.09)‰, and at this point, the carbon stable isotope ratio of DGP shows dilution effect during the liquefaction process, and the dilution value is -1.90‰. When the liquefaction temperature is 160 ℃, the δ¹³C value of DGP increases from (-15.66±0.17)‰ to (-11.95±0.09)‰, while the δ13C of DGP increases from (-18.09±0.16)‰ to (-9.26±0.06)‰ at 180 ℃, the carbon stable isotope enrichment values are 3.71‰ and 8.83‰, respectively. Meanwhile, the enrichment factor at 140, 160 and 180 ℃ are (-0.44±0.05)‰ (R² is 0.953 2), (0.17±0.01)‰ (R² is 0.981 6), (0.32±0.03)‰ (R² is 0.959 2), respectively. And combining with the variation tendency of the DGP’s yield, all of the above results show that, C—O, C—H bonds just participate in reacting in the process of glucose to generate DGP by dehydration. But during the generation of ethyl levulinate from DGP by decarboxylation and rearrangement isomerization reactions, there are not only breaking and forming C—O, C—H bonds, but also involving C—C, which leads to the latter having more significant carbon isotope kinetic effect. Therefore, it is speculated that the DGP directly generates levulinic acid through further decarboxylation and isomerization without undergoing 5-hydroxymethylfurfural. The results show that CSIA technology can effectively study reaction paths in complex systems such as glucose catalytic liquefaction and provide more information for liquefaction mechanism.