Abstract: In order to evaluate the temporal-spatial characteristics and risk assessment of Ampicillin (AMP) in the water source of Nanjing Section of the Yangtze River, 14 sampling sites of Nanjing section of Yangtze River were sampled in November, December 2019 and January, 2020, respectively, and AMP in water was analyzed by SPE-UPLC-MS/MS. The results show that the total mass concentration of AMP ranged from ND to 42.44 ng·L^-1, the average mass concentration was 19.67 ng·L^-1, and the detection rate was 57.5% after testing the samples collected from 14 sampling sites for three consecutive months. The detection rate of AMP in three months was 7.14%, 100% and 66.67%, respectively. The detection concentration and detection rate of AMP in December and January were generally higher than that in November. Risk quotient method was used to assess the risk of AMP in water bodies. The results show that the ecological risk entropy Q_R of AMP in Nanjing Section of the Yangtze River ranged from 0.40 to 0.57, which was a moderate risk. AMP's health risk entropy Q_RH ranged from 3.2×10^-5 to 5.1×10^-5, which had no risk to human health.