Sixteen per- and polyfluoroalkyl substances (PFASs) were measured in liver (n = 52) and kidney (n = 18) tissues of Indo-Pacific humpback dolphins (Sousa chinensis) stranded in the Pearl River Estuary (PRE) of China between 2004 and 2016. The average concentrations of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and most of other PFASs in the liver samples were respectively greater than any records previously reported in cetaceans globally. PFOS levels in 46% of dolphin liver samples exceeded the hepatic toxicity threshold in cetaceans. For the first time, we found a U-shaped trend for the distribution pattern of perfluorinated carboxylic acids (PFCAs) between liver and kidney with increasing carbon chain lengths (C5-C16), whereas a descending trend (C4-C10) was found for perfluoroalkane sulfonic acids (PFASs), which may be explained by binding efficiencies of PFAS analogues to proteins. Dolphins with the highest levels of ∑PFASs (age-corrected) were clustered near the river outlets in Lingdingyang area, which agrees with the spatial distribution of PFASs in the environment. Significant temporal trends were observed for many PFASs. Concentrations of PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA) and perfluoroheptanoic acid (PFHpA) all peaked in year 2011, followed by a decreasing trend, while a consistently descending trend was shown for perfluoroundecanoic acid (PFUdA) and perfluorodecane sulfonate (PFDS). Our findings contribute to the knowledge of tissue distribution and spatiotemporal trends of PFASs in the PRE dolphins, which are valuable for us to understand the PFASs exposure risk and their industrial emission in Southern China.