Objective: To investigate the levels of mitosis regulatory enzyme polo-like kinase 1 (PLK1) and Aurora A in tumor tissues and serum in patients with extrahepatic cholangiocarcinoma, and their clinical significance. Methods: The expressions of PLK1 and Aurora A in 54 tumor specimens of extrahepatic cholangiocarcinoma and 20 specimens of tumor adjacent bile duct tissue were determined by immunohistochemical staining, and the serum concentrations of PLK1 and Aurora A in 25 patients with extrahepatic cholangiocarcinoma and 15 healthy subjects were measured by ELISA assay. The relations of their expressions with clincopathologic factors of the patients and the changes in their serum levels in the patients before and after operation were analyzed. Results: In patients with extrahepatic cholangiocarcinoma, the positive expression rates of both PLK1 and Aurora A were significantly higher than those in tumor adjacent bile duct tissue (66.7% vs. 25.0%; 63.0% vs. 15.0%, both P<0.05), and their expressions were all significantly associated with degree of tumor differentiation, TNM stage and lymph node metastasis (all P<0.05). The preoperative serum concentrations of both PLK1 and Aurora A were significantly higher than those in healthy control population (434.85 pg/mL vs. 256.00 pg/mL; 644.64 pg/mL vs. 375.73 pg/mL, both P<0.05), which were significantly declined after operation (all P<0.05). The expression levels of PLK1 and Aurora A in tumor tissue and their serum concentrations had significant consistency (r=0.55; r=0.64, both P<0.05), and either in tumor tissue or serum, there was a significantly positive correlation between the levels of PLK1 and Aurora (tumor tissue: r=0.47, P<0.01; serum: r=0.71, P<0.01). Conclusion: The levels of both PLK1 and Aurora A are increased in either tumor tissue or peripheral blood in patients with extrahepatic cholangiocarcinoma, their elevations are closely related to the malignant progression of extrahepatic cholangiocarcinom and their combined detection may have certain value for early diagnosis and estimating therapeutic effect and prognosis of extrahepatic cholangiocarcinoma.