Ethnopharmacological relevance: Glycyrrhiza uralensis Fisch (RC) and Coptis chinensis Franch (RG) are traditional Chinese medicines, which are classic drug pair in prescriptions to treat gastrointestinal diseases. Multi-herb therapy is one of the most important features of traditional Chinese medicine, but due to the complex components of herbal decoctions, the substances that actually exert their medicinal effects have not been fully elucidated. The discovery of Glycyrrhiza uralensis Fisch and Coptis chinensis Franch supramolecular parts (RC-RG SA) can provide a new perspective for explaining the mechanism of drug-pair compatibility.Aim of the study: The purpose of this study was to explore the active composition and identification of chemical constituents of RC-RG SA, and to explore the inhibitory effects of supramolecular parts on S. aureus and biofilm.Materials and methods: The micromorphology of RC-RG SA was characterized by SEM and DLS. Intermolecular forces between Glycyrrhiza uralensis Fisch and Coptis chinensis Franch determined by ITC. The chemical constituents of RC-RG SA were systematically analyzed by UPLC-ESI-MSn. The inhibitory effect of RC-RG SA on S. aureus was determined by turbidimetric method and plate coating method. The scavenging effect of RC-RG SA supramolecular parts on S. aureus biofilm were observed by MTT method, SEM and LSCM, respectively.Results: The microstructure of RC-RG SA was spherical with a particle size of 161.6 nm. ITC proved that the reaction between decoction of RC and RG was exothermic. A total of 70 compounds were preliminarily identified in RC-RG SA, including 34 flavonoids, 34 alkaloids and 2 triterpenoids. The inhibitory effect of RC-RG supramolecular parts on S. aureus proliferation and the ability to clear S. aureus biofilm were better than RC-RG codecoction and RC-RG non-supramolecular parts.Conclusions: The Glycyrrhiza uralensis Fisch and Coptis chinensis Franch co-decoctions' supramolecular components were an important substance that exerts its medicinal effect. Current study provided supramolecular strategies to reveal the active ingredients and the medicinal effect of the traditional Chinese medicine decoction.