Abstract: Chinese medicine supramolecular is an important material form and effective form of Chinese medicine compound. The form and activity of supramolecular are affected by many factors, and the mixing order is one of the key factors. In this study, dynamic light scattering (DLS), scanning electron microscopy (SEM), turbidimeter and Tyndall effect were used to observe the differences of micromorphology and physical properties of three different groups of Coptidis Rhizoma group + Gardeniae Fructus + Scutellariae Radix (CGS), Coptidis Rhizoma group + Scutellariae Radix + Gardeniae Fructus (CSG) and Scutellariae Radix + Gardeniae Fructus + Coptidis Rhizoma group (SGC). The main components of the three groups of decoction were analyzed by high performance liquid chromatography (HPLC). The antibacterial activity of the three groups of decoction was preliminarily compared by in vitro Staphylococcus model, and the dynamic formation process of supramolecules in the three groups of decoction was preliminarily discussed by molecular dynamics simulation (MDS) technology. The results showed that different mixing sequences led to changes in the morphology of supramolecular parts of Huanglian Jiedu Decoction, with significant differences in macroscopic phenomena, electron microscopy, particle size, potential and turbidity, and significantly affected the original antibacterial activity. In addition, the results of molecular dynamics simulation also show that the different mixing order of the main components of Huanglian Jiedu Decoction will significantly affect its self-assembly behavior. This study is to further explore the factors affecting the formation of supramolecular substance basis of Huanglian Jiedu Decoction, also provides a reference for the clinical rational administration of Huanglian Jiedu Decoction formula granules.