Accurate and high-throughput diagnostic methods for Clostridioides difficile infection (CDI) are essential for timely detection and effective clinical management. In this study, we developed recombinant single-domain antibody fragments (VHHs) targeting the major virulence factors of C. difficile, toxin A (TcdA) and toxin B (TcdB). Anti-TcdA and anti-TcdB VHHs were successfully expressed in Escherichia coli, yielding high purity and production efficiency. Using these VHHs, we established a rapid enzyme-linked immunosorbent assay (ELISA) system by optimizing antigen immobilization, blocking, antibody reaction, and washing conditions. The rapid ELISA demonstrated high sensitivity, with limits of detection in the nanogram range, comparable to those of conventional ELISA. The rapid ELISA completed the entire assay within four hours, whereas conventional ELISA required approximately 22 hours. The rapid ELISA system provided a simple workflow and practical advantages for the development of ready-to-use diagnostic platforms for detecting C. difficile.