Circulating cell-free DNA (cfDNA) in ampullary cancer patients was measured to clarify the correlation between cfDNA and clinicopathological factors and the impact of cfDNA on survival outcomes. Patients with ampullary cancer undergoing pancreaticoduodenectomy were included. Correlations between cfDNA and clinicopathological and prognostic factors were determined. The cfDNA levels in patients ranged from 1282 to 21,674 copies/mL, with a median of 6687 copies/mL. The cfDNA level was significantly higher in patients with lymph node involvement, lymphovascular invasion, abnormal serum carcinoembryonic antigen (CEA) level, and stage II and III cancer. Poor prognostic factors for ampullary cancer included high cfDNA > 6687 copies/mL, lymph node involvement, abnormal serum CEA > 5 ng/mL, and advanced stage II and III cancer. The 1-and 5-year survival rates were 92.0% and 66.5%, respectively, for patients with low cfDNA < 6687 copies/mL and 84.0% and 49.9%, respectively, for patients with high cfDNA > 6687 copies/mL (p < 0.001). After multivariate analysis, only the cfDNA level and stage were independent prognostic factors of ampullary cancer. Thus, the cfDNA level could act as a surrogate marker of both disease extent and biological aggressiveness of ampullary cancer. Moreover, cfDNA plays a significant role in the prognosis of resectable ampullary cancer.