Abstract: Fusarium graminearum is the main pathogenic fungus causing wheat head blight. It not only causes severe crop yield reduction, but also produces fungal toxins such as deoxynivalenol (DON) and 15-acetyl deoxynivalenol (15-ADON), which are dangerous food pollutants to people and livestock. For the genetic functional study of Fusarium graminearum, it is often necessary to culture mycelium on LTB medium in the laboratory to investigate its regulatory effect on DON and 15-ADON synthesis. However, the detection of DON and 15-ADON in the mycelium is focused on extracellular, with less focus on the detection of intracellular. Simultaneous detection of intracellular and extracellular DON and 15-ADON can provide more in-depth information of fungal toxin production and secretion functions. In the study, a method based on gas chromatography-mass spectrometry (GC-MS) for simultaneous detection of DON and 15-ADON was developed. The solvents for extracting DON and 15-ADON can be completely separated and the calibration curves of each extract show good linear relationship within a certain range. The mass spectrometric characteristic scan shows high ionization efficiency for both DON and 15-ADON. The retention time of DON and 15-ADON in chromatogram are 6.98 and 7.68 min, respectively. Qualitative analysis of DON was performed using m/z 295, 235, and 193 under single ion monitor (SIM) mode, while m/z 392, 235, and 193 were used for 15-ADON. The highest intensity ion m/z 235 was selected for the quantification of DON, and m/z 193 for the quantification of 15-ADON. This method was used to detect the intracellular and extracellular levels of DON and 15-ADON in wild-type PH-1 and tri5 mutant with DON biosynthesis deficiency. The intracellular DON and 15-ADON levels in PH-1 are determined to be (149.13±9.15) μg/g and (1833.31±185.33) μg/g, extracellular levels are (5910.35±468.23) μg/g and (45222.12±2726.81) μg/g, respectively. The study identified a mutant strain with the deletion of the first enzyme involved in the synthesis pathway of DON and 15-ADON, both DON and 15-ADON are not detected in the extracellular and intracellular of the tri5 mutant. Additionally, this research confirms that after 7 days of cultivation in LTB medium, the wild-type strain exhibits higher levels of 15-ADON in both intracellular and extracellular compared to DON, with extracellular levels of DON and 15-ADON being higher than intracellular levels. This method can provide a methodological reference for the functional study of toxin synthesis in Fusarium graminearum.