The human NOB1 gene is located at chromosome 16q22.1, encoding a protein consisting of one zinc ribbon domain. Here we aimed to efficiently generate a monoclonal antibody against NOB1 protein. We synthesized the peptide APVEHVVADAGAFLRH based on published NOB1 cDNA sequences. The peptide was chemically linked with the carrier protein keyhole limpet hemocyanin and then injected into Balb/c mice. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using either purified 6xHis-NOB1 fusion protein or the peptide. One MAb named H11 (IgG1), effective in detecting the native NOB1 protein, was characterized by ELISA and Western immunoblotting. By using the MAb, we found NOB1 protein was expressed in human lung, liver, spleen, and kidney tissues. Taken together, the MAb H11 would be helpful for understanding the distribution and functions of NOB1.