Helicobacter pylori is an important risk factor of duodenal ulcer (DU). Although many virulence factors of H. pylori have been identified, few have been reported to show an association with the pathogenesis of DU. The aims of this study were to identify H. pylori antigens showing a high seropositivity in DU and to develop a platform for rapid and easy diagnosis for DU. Since DU and gastric cancer (GC) are considered clinical divergent gastroduodenal diseases, we compared two-dimensional immunoblots of an acid-glycine extract of an H. pylori strain from a patient with DU probed with serum samples from 10 patients with DU and 10 with GC to identify DU-related antigens. Of the 11 proteins that were strongly recognized by serum IgG from DU patients, translation elongation factor EF-G (FusA), catalase (KatA), and urease alpha subunit (UreA) were identified as DU-related antigens, showing a higher seropositivity in DU samples (n = 124) than in GC samples (n = 95) (FusA: 70.2% vs. 45.3%, KatA: 50.8% vs. 41.1%, UreA: 44.4% vs 27.4%). In addition, we found that the use of multiple antigens improved the discrimination between patients with DU and those with GC, as the odds ratios increased from 1.82 (95% CI: 0.79-4.21, P = 0.1607) for seropositivity for FusA, KatA, or UreA alone to 4.95 (95% CI: 2.05-12.0, P = 0.0004) for two of the three antigens, and to 5.71 (95% CI: 1.86-17.6, P = 0.0024) for all three antigens. Moreover, a protein array containing the three DU-related antigens was developed to test the idea of using multiple biomarkers in diagnosis. We conclude that FusA, KatA, and UreA are DU-related antigens of H. pylori and the combination of these on a protein array provided is a rapid and convenient method for detecting serum antibody patterns of DU patients.