Gancao Fuzi decoction (GCFZT) is a traditional Chinese formula, which has been commonly used in clinical practice to treat inflammatory diseases. However, the active substance of GCFZT in the treatment of inflammation is not fully clarified. In this study, we used orthogonal experiments to design different GCFZT formulations, resulting in a total of 16 GCFZT formulations. Subsequently, UPLC-Q-TOF-MS/MS was used to analyze the chemical composition of different formulations, and the anti-inflammatory activity differences of these formulations were evaluated through an LPS-induced RAW264.7 inflammatory cell model. Combined with machine learning algorithms such as PLS-DA and RF, four main active substances in GCFZT were screened. Finally, network pharmacology techniques were used to investigate the potential anti-inflammatory mechanisms of these main active substances, and the results showed that GCFZT mainly regulates the expression of core targets such as ALOX5, NFKB1, and TLR4 through main active substances such as chlorogenic acid, riboflavin, and formononetin, thereby affecting the NF kappa B signaling pathway, the Toll-like receptor signaling pathway, and the Th17 cell differentiation. This study provides a reference for the anti-inflammatory mechanism of GCFZT and a scientific basis for its clinical application.