Perfluoroalkyl substances (PFASs) in source water is of growing concern for its adverse effects on human health and wildlife as well. The Yangtze River is the vital drinking water source in Jiangsu Province of China, but little attention has been paid on PFASs. The occurrence, spatial distribution and temporal trend of PFASs in 21 water sources along the Jiangsu section of the Yangtze River was investigated with sampling from 2018 to 2020. Moreover, health risk of PFASs was assessed by estimated intake dose and derived tolerable intake dose, while ecological risk was assessed by selected effect concentration and environmental exposure. PFASs concentrations in source water ranged from 12.0 to 128 ng/L, with perfluorooctanoic acid (PFOA) as the dominated congener. Fluorine chemical industry lead to a great increase of perfluorohexanoic acid (PFHxA) in its nearest water source. The estimated daily intake of PFASs through drinking was 0.54 and 0.82 ng/kg bw/day for adults and children. The major health risk was from perfluorooctane sulfonate (PFOS) and PFOA for their toxicity on liver, reproduction, development and immunity, with the maximum hazard quotient of 0.029 and 0.043 for adults and children in the worst scenario. The ecological risks from PFASs on nine species groups ranged from 2.7 × 10-10 to 5.2. PFOA and Perfluorobutane sulfonate (PFBS) were causing significant risk on wildlife, particularly on worms, mussels, and fish, which may further influence the structure and processes in the foodweb. Overall, PFASs, especially PFOS, PFOA and PFBS, induced considerable risk on human health and aquatic species in some hotspot area. It would be necessary to include them into monitoring in China and develop standards for different protection purposes.