Abstract: Glucose labeled with stable isotope ¹³C is a commonly used tracing tool, which have been applied in life sciences and other fields. In the evaluation of tracer tools, isotope abundance is a key indicator of tracer. In this paper, a method for the determination of ¹³C labeled glucose abundance based on gas chromatographymass spectrometry (GC-MS) technology is established. The isotope abundance of ¹³C labeled glucose was detected through the screening of derivatization reagents, the optimization of derivatization conditions and the selection of appropriate mass spectrometry acquisition mode. Through the preparation of different concentrations of glucose solution to explore the accuracy of the two acquisition modes, it was found that the accuracy of detection results can be greatly improved by using ion scanning mode (SIM) at low concentration. In addition, it was also found that the derivatization reagent N-methyl bis (trifluoroacetamide) (MBTFA) has a better derivatization effect than N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The calculated value of the established ¹³C labeled glucose detection method was basically consistent with the theoretical value, the relative standard deviations (RSD, n=8) were below 0.2%，and absolute deviation were below 0.3%. The method for isotope abundance detection is accurate and can provide ¹³C labeled glucose isotope abundance detection method and provide technical support for research in related fields.