Omega-3 polyunsaturated fatty acids (ω3-PUFA) are central to the growth and reproduction of aquatic consumers. Dissolved nutrients in aquatic ecosystems strongly affect algal taxonomic composition and thus the production and transfer of specific ω3-PUFA to consumers at higher trophic levels. However, most studies were conducted in nutrient-poor, oligotrophic lakes, leading to an insufficient understanding of how water nutrients affect algal ω3-PUFA and their trophic transfer in consumers in highly eutrophic lakes. We conducted a field investigation in a highly eutrophic lake and collected basal food sources (phytoplankton, periphyton and macrophytes) and aquatic consumers (invertebrates, zooplankton and fish), and measured their fatty acid (FA) composition. Our results showed that periphyton and phytoplankton were both important sources of ω3-PUFA supporting the highly eutrophic lake food web. High water nutrient levels led to low ω3-PUFA levels in phytoplankton and periphyton, resulting in decreased nutritional quality. Consequently, ω3-PUFA of invertebrates and zooplankton reflected variations in ω3-PUFA of phytoplankton and periphyton, respectively. The ω3-PUFA levels of fish decreased as phytoplankton and periphyton ω3-PUFA decreased. Among fish, the Redfin Culter (Cultrichthys erythropterus) and Bar Cheek Goby (Rhinogobius giurinus) exhibited significantly higher levels of EPA and DHA compared to the Pond Loach (Misgurnus anguillicaudatus), which may have been caused by their different feeding modes. Decreases in the ω3-PUFA levels of basal food sources may be one of the causes leading to the reduction of trophic links in aquatic food webs. Our study elucidated the sources and fate of ω3-PUFA in highly eutrophic lakes, complemented previous studies in oligo- and mesotrophic lakes, and emphasized the role of high-quality food sources. Our results offer new perspectives for the conservation and management of highly eutrophic lake ecosystems.