Due to the lack of appropriate tumor markers with optimal cut-off values to predict the prognosis of gallbladder cancer (GBC), this study aimed to demonstrate the relationship between prognosis and the levels of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), and to determine optimal thresholds. In total, 539 patients diagnosed with GBC were examined. The relationship between tumor marker levels and overall survival (OS) was analyzed. The C-tree method was used to suggest tumor marker thresholds, and multivariate analysis was conducted to identify prognostic factors for overall survival. The mean age of the patients was 65.3 years, and the 5-year overall survival rate in all patients was 68.9%. Following the C-tree method, the optimal cut-off value was set at 5 IU/mL for CEA and at 65 IU/mL for CA 19-9. Multivariate analysis revealed that age, CA 19-9 level, operative method, T stage, and N stage were significant prognostic factors for OS. Consequently, CA 19-9 had a stronger association with prognosis than CEA, and 65 IU/mL for CA 19-9 may be suggestive in evaluating the prognosis of GBC. Moreover, it could be an effective indicator for determining the surgical extent necessary and the need for adjuvant treatment.