Garlic oil was extracted from garlic and qualitatively analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). Using enzyme activity real-time monitoring technology， it was revealed that Garlic oil， diallyl disulfide (DADS) and diallyl trisulfide (DATS) had partial inhibitory effects on glucose 6 phosphate dehydrogenase (G6PD) activity， and the inhibition was a mixed inhibition of competitive and non-competitive by enzymatic reaction kinetics； Molecular fluorescence technique analysis further showed that DADS/DATS had static quenching effect on G6PD， and there was a binding site for DADS/DATS and G6PD protein； Thermodynamic analysis revealed that the DADS-G6PD system was mainly combined through hydrophobic interactions， and the DATS-G6PD system was mainly combined through hydrogen bond and van der Waals force； 3D molecular docking and 2D molecular docking techniques further confirmed that DADS/DATS could enter the G6PD molecule， and the binding performance of DADS and G6PD was better； DADS could form hydrophobic interactions with Ala-377， Val-376， Phe-216， Gly-222， Phe-221， Pro-223 and other hydrophobic amino acid residues around the G6PD binding site， and interacted with Arg-219， Asn-218 ， Lys-275， Arg-215， Trp-225 and other polar amino acid residues formed electrostatic attraction， and hydrophobic interactions were dominant. DATS could form hydrophobic interactions with Phe-253， Gly-254， Ala-335， Ile-472， Ile-255， Leu-469， Leu-305 and other hydrophobic amino acid residues around the G6PD binding site， and interacted with Arg-175， Lys-476， Arg-257， Thr-334， Thr-333， Glu-473 and other polar amino acid residues formed electrostatic attraction. In addition， S in the center of DATS could form a hydrogen bond with Phe235 of G6PD， with a bond length of 3.2 ?魡. Hydrophobic interaction， electrostatic force and hydrogen bonding were the main modes of action of DADS/DATS on G6PD.