Monoclonal antibodies (McAbs) against human liver DnaJ-like protein (HLJ1) was produced by using lymphocyte-hybridoma technique and then one method for the detection of HLJ1 antigen was established. Two hybridoma cell lines which stably secreted monoclonal antibodies against HLJ1 were generated and named for A4C7 and C4C8. Subtypes of the two McAbs were both IgG1, and the antibodies showed high titer and good specificity. Using the prepared monoclonal antibody, human embryonic liver tissues were examined by immunohistochemistry. The results indicated that HLJ1 located in the cytoplasm of the human embryonic liver cell. A double antibodies sandwich ELISA was established by using C4C8 and HRP labeled A4C7. This assay had good specificity, and the lowest detection limit was 7.5 ng/mL and the linear range was 7.5-750 ng/mL. In conclusion, an immunohistochemistry method and a sensitive sandwich ELISA were established for the detection of HLJ1 protein.