The behavior and fate of PFOS (perfluorooctanesulfonate) in the aquatic environment have received great attention due to its high toxicity and persistence. The nanoscale supramolecular mechanisms of interaction between PFOS and ubiquitous EPS (exopolymers) remain unclear though EPS have been widely-known to influence the bioavailability of PFOS. Typically, the exposure patterns of PFOS in aquatic animals changed with the EPS-PFOS interaction are not fully understood. This study hypothesized that PFOS exposure and accumulation pathways depended on the PFOS-EPS interactive assembly behavior and animal species. Two model animals, zebrafish and chironomid larvae, with different feeding habitats were chosen for the exposure and accumulation tests at the environmental concentrations of PFOS in the absence and presence of EPS. It was found that PFOS triggered the self-assembly of EPS to form large aggregates which significantly trapped PFOS. PFOS accumulation was significantly promoted in zebrafish but drastically reduced in chironomid larvae because of the nanoscale interactive assembly between EPS and PFOS. The decreased dermal uptake but increased oral uptake of PFOS by zebrafish with large mouthpart size could be ascribed to the increased ingestion of PFOS-enriched EPS aggregates as food. For the chironomid larvae with small mouthpart size, the PFOS-EPS assemblies reduced the dermal, oral and intestinal uptake of PFOS. The nano-visualization evidences confirmed that the PFOS-enriched EPS-PFOS assemblies blocked PFOS penetration through skin of both animals. These findings provide novel knowledge about the ecological risk of PFOS in aquatic environments.