Background: Obstructive jaundice is one of the most common symptoms which can be caused by both malignant and benign hepato-biliary-pancreatic diseases. The differences and the differential diagnostic roles of the serum CA19-9, total bilirubin (TBIL) and especially, the ratio of CA19-9 to TBIL in these patients have not been well elucidated. This study compared the differences and the differential diagnostic roles of the increase-folds of the serum CA19-9, TBIL and the ratio of increase-folds of CA19-9 to increase-folds of TBIL in 508 cases of malignant (MOJ) and benign (BOJ) obstructive jaundice patients. Methods: Totally, 508 cases of obstructive jaundice patients with pathological results were retrospectively enrolled. 342 cases were MOJ, including gallbladder adenocarcinoma, extrahepatic cholangiocarcinoma, periampullar adenocarcinoma and pancreatic adenocarcinoma.The other 166 cases were BOJ, including cholelithiasis, primary sclerosing cholangitis (PSC), IgG4 related sclerosing cholangitis (IRSD), inflammatory stricture of bile duct, and adenoma. The data of the increase-folds of serum CA19-9, TBIL and the ratio of increase-folds of CA19-9 to increase-folds of TBIL was collected and analyzed. The ROC (receiver operating characteristic) curve was adopted to determine the optimal cutoff value to evaluate their differential diagnostic roles. Results: The CA19-9 was elevated in 94.15% of the MOJ, compared to 67.10% in BOJ (P<0.0001). The increase-folds of the CA19-9 (76.52±15.04 vs 3.55±0.41, P< 0.0001) and the increase-folds of TBIL (7.10±0.25 vs 5.95±0.36, P=0.0034) and the ratio (14.96±5.05 vs 0.79±0.93, P=0.0026) in MOJ were significantly higher than BOJ. The ROC curves indicated that the TBIL showed minimal differential diagnostic power (AUC=0.590), the increase-folds of CA19-9 and the ratio showed stronger differential diagnostic power (AUC=0.815 and AUC=0.889, respectively). The optimal value of the increase-folds of serum CA19-9 and the ratio for differential diagnosis was 4.5 and 0.88, respectively. Further, the combination of the increase-folds of serum CA19-9 and the ratio could increase the specificity and accuracy of the diagnosis of MOJ. Conclusion: The serum CA19-9, TBIL and the ratio in MOJ and BOJ are significantly different. The ratio of the increase-folds of CA19-9 to increase-folds of TBIL has stronger differential diagnostic roles than the increase-folds of CA19-9 or TBIL alone. The combination of the increase-folds of the CA19-9 and the ratio can further improve the specificity and accuracy of the diagnosis of MOJ.