Diallyl trisulfide (DATS) has shown various biological activities, such as antimicrobial, antihyperlipidemic, antithrombotic, and anticancer effects. It is a highly water insoluble thioether compound, which irritates blood vessels and degrades rapidly under normal conditions. Hence, a suitable DATS delivery system and the related stability studies are both valuable for the clinical use of DATS. The DATS micellar injection in the present study was prepared using propylene glycol, alcohol, Tween 80, and water by the self-assembling micellar method, which effectively avoided irritation and degradation of DATS. The high-pressure liquid chromatography-UV assay method was established and validated for the quantitative determination of DATS micellar injection. The major degraded compounds were identified as diallyl disulfide and diallyl tetrasulfide by gas chromatography-mass spectrometry. 3-ethenyl-3,6-dihydro-1,2-dithiin and 3-ethenyl-3,4-dihydro-1,2-dithiin might be the thermal decomposed products of DATS in process of gas chromatography-flame ionization detector analysis. The main factor influencing stability in DATS micellar injection was ascertained to be temperature. This investigation might be a beneficial contribution to further identification of DATS-degraded products and exploration of the delivery system of DATS.