Abstract: Traditional Chinese medicine decoction is a polydisperse system composed of multiple phases. On the basis of phase separation, conducting chemical composition analysis and efficacy evaluation are helpful to clarify the transformation rules of the effective substances of traditional Chinese medicine. In this study, the classical prescription, Ling-Gui-Zhu-Gan Decoction was used as the object to track the real-time change of the particle size of the decoction during the boiling process; different phases were separated by centrifugation and ultrafiltration, and various types of chemical components were analyzed. The therapeutic effect of the decoction and its representative phase was evaluated by establishing an in-house mouse obstructive kidney model. The results showed that the mixing degree of particle size distribution decreased during the boiling process of Ling-Gui-Zhu-Gan Decoction, and finally stabilized around 200 nm. After phase separation, small molecules, polysaccharide, protein, lipids, RNAs and other components were mainly distributed in the colloidal phase. The whole decoction and the colloid phase showed almost equal activity in alleviating the process of obstructive renal fibrosis. This investigation further confirmed that the chemical components of different Chinese herbs were redistributed and aggregated in the colloidal phase during the boiling process of Ling-Gui-Zhu-Gan Decoction, which became the main form of the whole decoction to play its therapeutic effect. This provides a basis for understanding and exploring traditional Chinese medicine from the perspective of phase state. The animal experiment has been approved by the Animal Experiment Ethics Committee of Nanjing University of Chinese Medicine (No: 202501A012).