Human arrest defective 1(hARD1) is an acetyltransferase catalyzing the N-terminal acetylation of proteins after translation. The high expression of hARD1 could be an indicator of the breast cancer. In current study, we produced an anti-hARD1p monoclonal antibody that could specifically recognize ARD1 in breast cancer tissues by using the immunohistochemical assay. The full-length His-tag hARD1 protein (1~235 aa) was over-expressed in Escherichia coli, and purified recombinant protein was injected into Balb/c mice to perform immunization procedure. Eight stable positive monoclonal cell lines were isolated. ELISA results demonstrated that all light chains of antibodies were κ, and the heavy chains displayed three subtypes IgG1, gG2a and IgG2b, respectively. A monoclonal antibody, which could specifically recognize hARD1 protein in breast cancer tissues, was identified by screening different cancer tissues using antibody-specificity method. Further, the specificity of the antibody was confirmed by Western blotting analysis. Our study would facilitate breast cancer diagnosis by using this ARD1 monoclonal antibody in clinic. Also, this antibody could be used as an important tool for further investigating the role of ARD1 in tumorigenesis.