To immunize mouse with KLH-conjugated synthetic polypeptide fragment (C14) of cytokeratin 7 (CK7) against the dominant epitope, after cell fusion and being screened, we prepared monoclonal antibodies (mAbs) against CK7, collected clinical tissues for immunohistochemical identification. The results showed that C14 polypeptide has significant immunoreactivity and immunogenicity as an antigen, and three diagnostic monoclonal antibody cell lines that stably secretes antibodies have been obtained. The 7A8 monoclonal antibody with better affinity was selected for immunohistochemical identification, and the antibody concentration was 0.49 μg/mL which is lower than 2～5 μg/mL in the commonly used CK7 detection kits in hospitals, showing high sensitivity. It showed a good specific reaction with the collected clinical specimens, and the key raw material were provided by the screened 7A8 monoclonal antibody for the development of CK7 rapid diagnostic reagents.