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Perfluorooctane sulfonate (PFOS) is an accumulative emerging persistent organic pollutant that coexists with copper (Cu) in aquatic environments and is a severe threat to human health. PFOS has genotoxicity, male reproductive toxicity, neurotoxicity, developmental toxicity and endocrine disrupting effects, and is considered to be a kind of environmental pollutant with systemic multi-organ toxicity. However, the combined effects of PFOS and Cu on aquatic organisms have not been analyzed previously. In this study, we investigate the response of Vallisneria Natans (V. natans) and biofilms to single and combined treatments of PFOS and Cu at environmental concentrations (1.0, 10.0, and 100.0 μg·L−1). Results indicate that single and combined treatment triggered the reactive oxygen species (ROS) metabolism with increased activities of GSH, MDA, CAT, TSOD and an antagonistic joint toxicity. Meanwhile, transcriptomic analyses showed that the peroxidase (PER), 9 glutaredoxin (GRX), and 39 glutathione S-transferase (GST) expression of genes related to ROS detoxification was up-regulated. In addition, single and combined exposure induced the up-regulation of the transport channel genes which encode aquaporins, rapid-type anion channels, and copper-exporting P-type ATPase transporters, promoting the uptake of PFOS and Cu in V. natans , Enhancing detoxification and mitigating the effects of Cu and PFOS exposure. Moreover, PFOS and Cu changed the structural characteristics of extracellular polysaccharides in biofilms, synthesizing large amounts of β- D -glucopyranose PS, α- D -glucopyranose PS. And the community diversity and species evenness changed, with Rhizobiaceae , Chloroplast , and Weeksellaceae being the dominant phylum. This study provides insights into the molecular mechanism of joint toxicity of PFOS and Cu by Transcriptomics and PCR analysis on the submerged macrophyte and their periphyton biofilms. [Display omitted] • Toxicity of PFOS and Cu mixture on V. natans was antagonistic. • PFOS and Cu triggered the transporter-mediated passive process. • β- D -glucopyranose PS was the dominant EPSs in biofilms with Cu exposure. • PFOS triggered more α- D -glucopyranose PS generated in biofilms. • GSEA was first reported for analysis of the joint toxicity in submerged macrophyte. [ABSTRACT FROM AUTHOR]