Objective: To determine the diagnostic value of the combination detection of the tumor markers, CA50, CA125, CA242, CA19-9 and CEA for hilar cholangiocarcinoma. Methods: Ninety patients admitted to our hospital in the recent two years, who were confirmed having hilar cholangiocarcinoma by postoperative pathology, were included in the observation group, and another group of 91 patients with benign biliary tract diseases hospitalized during the same period served as control group. The serum levels of CA50, CA125, CA242, CA19-9 and CEA of the two groups were detected by automatic electrochemiluminescence analyzer, and the sensitivities, specificities and accuracies of the five tumor markers of the two groups were determined, respectively. Results: The serum levels of CA50, CA242, CA19-9 and CEA in the observation group were all significantly higher than those of the control group, (all P<0.01), but the difference of the serum levels of CA125 between the two groups had no statistical significance (P>0.05). In hilar cholangiocarcinoma, the serum CA19-9 showed the highest positive rate (86.67%) among the five markers, followed by CA242 (63.33%), and CA50 (60%), successively. Between the two groups, the positive rates of the five tumor markers, except for CA125, had significant differences (all P<0.05). For the diagnosis of hilar cholangiocarcinoma, serum CA19-9 had the best sensitivity (93.98%), while CEA had the best specificity (94.60%). Conclusion: Combination detection of CA50, CA242, CA19-9 and CEA is helpful for differential diagnosis of hilar cholangiocarcinoma and benign biliary tract diseases.