Background <p>Carbohydrate antigen 19-9 (CA19-9) represents an established prognostic biomarker for biliary tract cancers (BTCs). Clinical utility remains limited among patients with Lewis antigen negativity or incomplete fucosyltransferase 3 (<i>FUT3</i>) gene function, conditions associated with absent or reduced CA19-9 production. Duke pancreatic monoclonal antigen type 2 (DUPAN-2), a biosynthetic precursor of CA19-9, shows compensatory elevation under such conditions. This study evaluated the prognostic significance of the combined preoperative assessment of CA19-9 and DUPAN-2 among patients with resectable BTCs.</p> Patients and Methods <p>Patients who underwent curative-intent surgery for BTCs between 2009 and 2022 were included. Eligible patients were classified into four groups according to preoperative biomarker levels: normal CA19-9/normal DUPAN-2 (N/N), normal CA19-9/high DUPAN-2 (N/H), high CA19-9/normal DUPAN-2 (H/N), and high CA19-9/high DUPAN-2 (H/H). Clinicopathological features, overall survival (OS), and recurrence-free survival (RFS) were compared. Multivariate analyses identified independent prognostic factors.</p> Results <p>The cohort included 276 patients with BTCs who underwent curative resection, comprising 41 intrahepatic cholangiocarcinomas, 72 hilar cholangiocarcinomas, 57 distal cholangiocarcinomas, 53 gallbladder carcinomas, and 53 ampullary carcinomas. Age and tumor distribution showed no significant intergroup differences. The N/N group demonstrated significantly superior OS and RFS (<i>P</i> &lt; 0.001). Multivariate analysis identified the N/N profile as an independent favorable prognostic factor for OS and RFS (<i>P</i> &lt; 0.001).</p> Conclusions <p>Concurrent normal CA19-9 and DUPAN-2 levels independently predicted favorable outcomes. Combined preoperative biomarker assessment may contribute to prognostic stratification of BTCs, including among patients with Lewis antigen negativity or impaired FUT3 function.</p>