Abstract: This study initially explored the influence of different brewing methods of Rhei Radix et Rhizoma-Coptidis Rhizoma formula granules on the "bitter and cold" bias (formula granule-cold mixing, FG-CM; formula granule-hot mix and reheating, FG-HMR). We characterized the differences in morphology, molecular thermodynamics, spectroscopy and diarrhea phenotype of mice of the two brewing methods decoctions, in order to analyze the potential impact of different brewing methods on the clinical efficacy of Rhei Radix et Rhizoma-Coptidis Rhizoma formula granules. The research found that there were a large number of flocs in the FG-CM system under macroscopic observation, and the FG-HMR system was in a uniform turbid state. Similarly, under the scanning electron microscope, the particle size of FG-HMR was more uniform and the scale was smaller. The ITC experiment found that the interaction between the formula particles of Rhei Radix et Rhizoma-Coptidis Rhizoma was obvious. However, the characterization results of UV-vis, IR and HPLC indicated that the chemical types and contents of FG-CM and FG-HMR were basically consistent, suggesting that the brewing method did not change the chemical composition but there was molecular interaction. Through the mouse diarrhea model, it was found that compared with the FG-CM group, the "bitter and cold" bias in the FG-HMR group was more moderate. It could better maintain the normal intestinal homeostasis and intestinal tissue structure of mice, reduce the rate of soft stools in mice, and alleviate the side effects of diarrhea in mice. This study started from the perspective of material basis and diarrhea phenotype, with the expectation of providing a reference for the clinical administration of formula granules containing Rhei Radix et Rhizoma-Coptidis Rhizoma. The animal experiment was approved by the Ethics Committee of Beijing University of Chinese Medicine (Approval No.: BUCM2024-093-M).