NOB1, a transcription-associated protein, may play important roles in the development of many cancers. In this study, we have efficiently generated one monoclonal antibody (MAb) against NOB1. Enzyme-linked immunosorbent assay (ELISA) and western blot were used to screen the hybridomas. As a result, one MAb named L6 (IgG1) effective in detecting the recombinant and the cellular NOB1 protein was characterized. Using L6, NOB1 was found to be upregulated in gastric cancer cells and tissues compared with normal gastric epithelial cells and nonneoplastic tissues. The expression of NOB1 was also found to be higher in multidrug-resistant gastric cancer cells than that of sensitive cells. This novel MAb will be valuable for investigating the role of NOB1 in carcinogenesis and multidrug resistance of gastric cancer.