The focus of this study is to explore the impact of gut microbiota in different states on the blood components of couplet medications (dried toad skin and radix clematidis) and to identify drug metabolites associated with the gut microbiota. By constructing a pseudo-sterile rat model and combining non-targeted metabolomics with plasma pharmacology, we found that the plasma metabolites of couplet medications underwent significant changes in different gut microbiome environments. The GABA and PGE1 levels in the model group and the model+TCM (traditional chinese medicine) group were both significantly lower than those in the normal+TCM group. When the gut microbiota is imbalanced, drug interventions cannot significantly increase the levels of GABA and PGE1. It further confirmed the correlation between the levels of GABA and PGE1 and the gut microbiota. Based on the results of non-targeted metabolomics, we applied network pharmacology and molecular docking to explore the core targets for colorectal cancer treatment based on gut microbiota. In the end, we identified TNF and PPARG as the two core targets. These research findings provide a possibility for clarifying the molecular mechanisms of couplet medications in the treatment of colorectal cancer. It also laid the foundation for further clarifying the molecular mechanisms of Chanling Paste in the treatment of colorectal cancer.