Presently, no effective markers are available to facilitate gallbladder cancer (GBC) diagnosis. This study aims to explore available markers for GBC diagnosis. Clinical data of 144 GBC and 116 cholelithiasis patients were retrospectively reviewed. Logistic regression analysis was performed to evaluate GBC risk factors. A receiver operating characteristic (ROC) curve was used to assess the diagnosis value of the risk factors. By comparing the characteristic of GBC and cholelithiasis patients, the following factors exhibited statistical difference: age, gender, gallstones, total bilirubin (TB), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), platelet count (PLT), CA125 (carcinoembryonic antigen 125), and CA199 (carbohydrate antigen 199). Logistic regression analysis indicated that age [odds ratio (OR), 1.032; 95%confidence interval (95% CI), 1.004 to 1.061; P = 0.024], gender (OR, 0.346; 95% CI, 0.167 to 0.716; P = 0.004), gallstones (OR, 0.027; 95% CI, 0.007 to 0.095; P < 0.001), ALP (OR, 1.003; 95% CI, 1.000 to 1.006; P = 0.032), TB (OR, 1.004; 95% CI, 1.000 to 1.009; P = 0.042), and CA125 (OR, 1.007; 95% CI, 1.002 to 1.013; P = 0.011) were independent risk factors for GBC. According to the ROC curve, CA125 [area under curve (AUC), 0.720], ALP (AUC, 0.713), TB (AUC, 0.636), and age (AUC, 0.573) were valuable diagnosis markers. Additionally, based on the independent risk factors, the GBC diagnosis model was established. Age, TB, ALP, and CA125 can be used as auxiliary diagnosis factors of GBC. The diagnosis model provides a quantitative tool for GBC diagnosis when comprehensively considering various risk factors.