Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), HMab-119 (IgG, kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. HMab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although HMab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with HMab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that HMab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.