Stream ecosystems have been impacted by multiple anthropogenic stressors including the loss of riparian canopy cover. However, it remains unclear how these stressors shape the profiles and transfer of fatty acids in basal resources, and subsequently affect the trophic linkages in stream food webs. We collected 158 food web samples including three basal resources (periphyton, fine benthic organic matter (FBOM) and seston), two primary consumers (macroinvertebrate and omnivorous fish), and one secondary consumer (carnivorous fish) together with water quality samples across an anthropogenic disturbance gradient in a small catchment (Laoguan River) of the Yangtze River, China. We investigated the influence of riparian land-use changes, canopy cover and water quality on the nutritional quality indicated by the proportions of eicosapentaenoic acid (EPA) in periphyton, seston and FBOM and their transfer in stream food webs. Our results showed that land use primarily affected the reduced the proportions of EPA in periphyton more than seston and FBOM. It was confirmed that loss of canopy coverage reduced the proportions of EPA of periphyton due to the significant correlations and difference between two groups: the least disturbed group (forest > 90 %) and the highly disturbed group (high agriculture/urban). Compared to the least-disturbed sites, food webs appeared to be simpler with less trophic linkages at highly disturbed sites affected by agricultural and urban land uses. Finally, we validated that EPA was the important linkage between periphyton and primary consumers-macroinvertebrates, and further second consumers- omnivore fish, which may account for the decrease of network of trophic links at highly disturbed sites. This study provides insights into how anthropogenic stressors, particularly land-use changes and loss of riparian canopy cover, affect the nutritional quality of basal resources and simplify food web structures in stream ecosystems, highlighting the critical role of eicosapentaenoic acid (EPA) in linking trophic levels.