Urechis unicinctus is a marine benthic invertebrate that relies primarily on humoral immunity within the nonspecific immune system for body defense. In order to elucidate the protein components of the coelomic fluid and investigate its immune response mechanism, proteomic analysis and antimicrobial characterization of the coelomic fluid of U. unicinctus were carried out. A total of 2194 proteins were identified, with 427 showing differential expression in coelomocytes compared to those in the coelomic fluid supernatant. Of these proteins, 346 were upregulated and 81 were downregulated. Next, these identified proteins were analyzed for biological information, including GO, COG, and KEGG pathway analysis. The results from the GO analysis revealed that cytoplasm and ATP-binding were the two prominent categories of proteins found in the coelomocytes as well as the coelomic fluid supernatant of U. unicinctus. From the COG analysis, it was evident that the categories of proteins identified in the coelomocytes were essentially the same as those identified in the coelomic fluid supernatant, with only the number of proteins differing. The KEGG pathway analysis indicated that 45 pathways from the coelomic fluid supernatant and 42 from the coelomocytes were profiled, with carbon metabolism and ribosome being the two most prominent pathways. The Pfam database displayed that the immune-related proteins in U. unicinctus were neurofascin, cell adhesion molecule 4, receptor-type tyrosine-protein phosphatase F, limbic system-associated membrane protein, four and a half LIM domains protein 2, neuroglian, fasciclin-2, and neural cell adhesion molecule. Furthermore, the active substances from the coelomic fluid underwent isolation, purification, and antimicrobial characterization. The process yielded two purified components (b