Hepatocellular carcinoma (HCC) is a malignancy that is associated with high mortality rates in Asia. These tumors are highly invasive and their etiology is frequently unknown. Thus, most patients are diagnosed in the middle and late stages of the disease, and thus do not have sufficient time for therapy. Therefore, it is essential to study the early diagnosis and treatment of HCC; in this regard, the study of tumor‐associated antigens has received much attention. Here, antigens from the human primary HCC cell line, QGY‐7703, were used to immunize mice in order to prepare monoclonal antibodies. The specific antigen recognized by antibody 11C3 was purified from total protein lysates of QGY‐7703 by immunoaffinity chromatography. The validity of the candidate antigen as a new HCC‐associated marker was tested using SDS/PAGE, western blot, HPLC‐ESI‐MS/MS, and RT‐qPCR. Our results showed that the levels of CK10 in HCC‐derived cell lines were significantly higher than those in normal liver cells. Thus, we suggest that CK10 may be involved in the formation and development of HCC, and may be a therapeutically targetable tumor‐associated antigen.