Per- and polyfluoroalkyl substances (PFAS) raise significant concerns due to their persistence, bioaccumulation potential, and toxicity to both ecosystems and human health. However, the long-term trends of PFAS in aquatic environments remain inadequately explored. In this study, we systematically assessed the spatiotemporal distribution, periodic fluctuations, source apportionment, and risk evaluation of 12 PFAS in the Rhine River based on the long-term measuring data collected from 2007 to 2019. The study revealed that the mean concentration and mass flux of total PFAS during this period were 32.83 ng L−1 and 6.36 × 104 μg s−1, declining at an annual rate of 3.70% and 3.82%, respectively. Wavelet analysis demonstrated that the most prominent periodic oscillation of PFAS was 40–60 months. Regarding the sources of PFAS, we employed the self-organizing map (SOM) and the positive matrix factorization (PMF) model for source apportionment. The results indicated that the primary sources of PFAS were agrochemical, pharmaceutical and textile industries, accounting for 38.1% of the total concentration. The contribution from household contamination, tannery industry, and coating materials has increased annually. In contrast, the share of electrochemical fluorination and chemical recycling has shown a continuous decline. The risk quotient (RQ) and hazard quotient (HQ) calculations for three age groups indicated that PFAS exposure did not pose a significant risk to ecological or human health. Implementing source-oriented mitigation strategies is crucial to effectively reduce the ecological and human health risks of PFAS in receiving waters.