Monitoring plant biodiversity in large river systems is essential for ecological assessment but remains logistically challenging due to poor visibility, strong currents, and broad spatial scales. Environmental DNA (eDNA) metabarcoding offers a minimally invasive solution, allowing large-scale biodiversity surveys by analysing aquatic and terrestrial plant diversity from river water samples. Here, we present the first application of multi-marker eDNA metabarcoding for integrated plant biodiversity assessment in a large river system, targeting aquatic vegetation, invasive species, and land-use-associated flora along the Upper Rhine River between Basel (Switzerland) and Strasbourg (France). Our method recovered 24 aquatic plant species (18% of reads), underscoring the potential of this approach for monitoring macrophytes in large rivers where conventional visual surveys are impractical. Land-use indicators, including crop and ornamental species, comprised 5.5% of reads, and contrasted between the human-modified canal of the Rhine and the natural river channel. We also detected 16 exotic plant species, highlighting the method’s potential for monitoring and managing invasive populations. However, the spatial resolution of species detection could benefit from closer sampling along the river and additional samples across its cross-section. This study highlights the utility of multi-marker eDNA metabarcoding as a scalable, non-invasive tool for catchment-wide vegetation monitoring and environmental management in large river systems.