Abstract: Objective To evaluate the diagnostic value of multiple tumor markers protein biochip (C-12) for patients with cholangiocarcinoma. Methods The serum levels of 12 tumor markers (TMs), including CA199, NSE, CEA, CA242, Ferritin, β-HCG, AFP, f-PSA, PSA, CA125, CA153 and HGH, were measured in 26 cholangiocarcinoma patients, 52 benign disease patients of digestive system and 65 normal controls. The most relevant TM and the combinations of TMs to the improvement of diagnosis were determined. Results The positive rates of CA199, CEA, CA242, Ferritin, AFP, CA125 and CA153 in the cholangiocarcinoma patients had significantly higher than those of the benign disease patients and normal controls (P<0.01). The serum levels of CA199, NSE, CA242, Ferritin, β-HCG, AFP, CA125, HGH and CA153 in the cholangiocarcinoma patients had significantly higher than those of the benign disease patients and normal controls (P<0.05~0.01). Among all the 12 TMs, the single best TM was CA199 and gave the sensitivity, specificity and validity of 69.2%, 96.9% and 89.0% for the cholangiocarcinoma, respectively.The best combination of 3 TMs (CA199+CA125+CEA) had not statistically significant comparied with C-12 and yielded a sensitivity of 91.7%, a specificity of 87.5% and a validity of 89.6%. Conclusions The multiple tumor markers protein biochip has a relatively high value in the diagnosis of cholangiocarcinoma. The combination detection of 3 TMs (CA199+CA125 +CEA) could replace 12 TMs of C-12 and may be the economical and effective in the diagnosis of cholangiocarcinoma.