The blood neutrophil-to-lymphocyte ratio (NLR) is reported to be a prognostic marker in several cancers. However, the prognostic role of NLR in patients with advanced cholangiocarcinoma on chemotherapy is unknown. A total of 221 patients with pathologically confirmed locally advanced or metastatic cholangiocarcinoma receiving first-line palliative chemotherapy were enrolled. Associations between baseline clinical and laboratory variables including NLR and survival were investigated. Patients were classified into two groups according to the NLR level (≤ 5 vs. >5). Median overall survival (OS) and time to progression (TTP) in patients with NLR ≤ 5 were 10.9 and 6.7 months, respectively, and 6.8 and 4.1 months in patients with NLR > 5 (P < 0.001, P = 0.002, respectively). In multivariate analysis, number of cycles of chemotherapy was a significant predictor of longer OS (HR 0.86, P < 0.001), whereas adverse prognostic factors for OS were CA 19-9 > 300 (HR 1.43, P = 0.025), CEA > 5 (HR 1.44, P = 0.029), higher stage (HR 1.69, P = 0.004), and NLR > 5 (HR 1.87, P < 0.001). NLR > 5 was also associated with reduced TTP (HR 1.66, P = 0.007). Among 50 patients with initial NLR > 5, 33 patients had NLR ≤ 5 after two cycles of chemotherapy and they had significantly better survival than the others (HR 0.48, P = 0.015). NLR independently predicts survival in patients with advanced cholangiocarcinoma undergoing chemotherapy. Considering cost-effectiveness and easy availability, NLR may be a useful biomarker for prognosis prediction.