Objective To observe the clinical efficacy and safety of circulating tumor cell (CTC) drug sensitivity-guided chemotherapy in the treatment of advanced biliary tract cancer. Methods Twenty-six patients with advanced biliary tract cancer treated in the Department of Oncology Biotherapy, Eastern Hepatobiliary Surgery Hospital, Naval Medical University (Second Military Medical University) from Jul. 2018 to Apr. 2020 were included. The CTCs were isolated and enriched from the peripheral blood. The drug sensitivity was tested with the fixed chemotherapy regimen commonly used in biliary tract cancer, and the best regimen was selected for chemotherapy (CTC group). Nineteen patients with advanced biliary tract cancer treated with GEMOX regimen (gemcitabine+oxaliplatin) during the same period were selected as the controls (GEMOX group). The objective response rate (ORR), disease control rate (DCR), progress-free survival (PFS), overall survival (OS) and safety of the 2 groups were observed. Results The efficacy and safety were evaluated in all the patients: 1 case achieved complete remission (CR), 4 cases achieved partial remission (PR), 12 cases had stable disease (SD) and 9 cases had progressive disease (PD) in the CTC group; and 1 case reached PR, 10 cases had SD and 8 cases had PD in the GEMOX group. The ORRs of the CTC group and GEMOX group were 19.2% (5/26) and 5.3% (1/19), and the DCRs were 65.4% (17/26) and 57.9% (11/19), respectively. The median PFS was 5.8 months (95% confidence interval[CI] 4.5-7.2 months) in the CTC group and 5.1 months (95% CI 4.6-6.4 months) in the GEMOX group, showing no significant difference (P=0.313). The median OS was 13.6 months (95% CI 10.8-16.3 months) in the CTC group and 7.9 months (95% CI 5.5-10.3 months) in the GEMOX group, showing significant difference (P=0.003). In all patients, the main adverse events were anemia, neutropenia, thrombocytopenia, fatigue and anorexia, all of which were tolerable, and there was no treatment-related death. Conclusion CTC drug sensitivity-guided chemotherapy improves the tumor response rate of advanced biliary cancers, prolongs the overall survival of the patients, and the adverse events are tolerable. CTC drug sensitivity test is convenient, and is valuable for accurate clinical selection of individualized chemotherapy regimens, so it is worthy of further promotion.