Bioaccumulation and human health risk assessment of Perfluoroalkyl acids (PFAAs) is important for pollutant hazard assessment. In this study, 26 aquatic organisms were collected from the Yangtze River estuary, the PFAAs concentrations in organisms were detected by liquid chromatography-mass spectrometry, and the trophic levels of organisms were constructed using nitrogen isotope analysis. The results showed that Perfluorobutane sulfonate (PFBS) was predominant in organisms with the mean concentration of 6.43 ± 8.21 ng/g ww. The biomagnification of organisms along the food chain was widespread, and the biomagnification factor (BMF) of perfluorooctane sulfonic (PFOS) was the most prominent. Trophic magnifcation factors (TMFs) of PFAAs were estimated in the marine food web, and TMFs >1 were observed in Perfluorodecanoic acid (PFDA), Perfluoroundecanoic acid (PFUnDA), Perfluorododecanoic acid (PFDoDA), and PFOS, indicating the biomagnifcation effects of these 4 individual PFAAs in organisms at Yangtze River estuary. The estimated daily intake (EDI) of PFBS was highest in adolescents aged 6–18 years, with EDIs of 18.9 ng/kg·bw/day for males and 14.0 ng/kg·bw/day for females. The hazard ratio (HR) of PFAAs reported in different age and gender groups were lower than 1.