Abstract: In colorectal cancer, tumor necrosis factor receptor-associated factor 2 (TRAF2) is a positive regulator of the Wnt/β-catenin signaling pathway and is closely related to the occurrence and development of colorectal cancer. This study aims to discover novel anti-colorectal cancer small molecules targeting TRAF2 by establishing a high-throughput TRAF2 affinity screening system. The study established and optimized a high-throughput TRAF2 screening system based on Dianthus, and conducted a large-scale screening of 3 716 traditional Chinese medicine (TCM) small molecules from a TCM small molecule library. Microscale thermophoresis (MST) and cellular thermal shift assay (CETSA) were further applied to verify the binding in both purified proteins and living cells. Among the candidates, the strongest binding compound, eupatilin (EUP), had an affinity of 7.87 μmol·L^-1 with TRAF2. TOPFlash assays showed that EUP can inhibit the activity of the Wnt/β-catenin signaling pathway and the growth of colorectal cancer cells and mouse small intestinal adenoma organoids. After TRAF2 was knocked out using CRISPR/Cas9 technology, the inhibitory effect of EUP on the Wnt signaling pathway and colon cancer cells was significantly weakened, indicating that TRAF2 is the functional target protein of EUP. In summary, the novel high-throughput screening method based on Dianthus constructed in this study can be used for the screening of small molecule targeted drugs, and it has been confirmed that EUP is a TRAF2-targeted small molecule that inhibits the development of colorectal cancer, providing a theoretical basis for the development of targeted TRAF2 anti-tumor drugs.