Environmental DNA (eDNA) extracted from water is routinely used in river biodiversity research, and via metabarcoding eDNA can provide comprehensive taxa lists with little effort and cost. However, eDNA-based species detection in streams and rivers may be influenced by sampling season, location, and other key factors such as water temperature and discharge. Research linking these factors and also informing on the potential of eDNA metabarcoding to detect shifts in ecological signatures, such as species phenology and functional feeding groups across seasons, is missing. To address this gap, we collected 102 water samples every two weeks for 15 months at a long-term ecological research (LTER) site and at three different positions in the river’s cross section, specifically the water surface, riverbed, and riverbank. We analyzed macroinvertebrate species and molecular Operational Taxonomic Unit (OTU) richness and temporal community turnover across seasons and sampling positions based on COI metabarcoding data. Using Generalized Additive Models, we found a significant influence of sampling season but not sampling position on community composition. Community turnover followed a cyclic pattern, reflecting the continuous change of the macroinvertebrate community throughout the year (‘seasonal clock’). Although water temperature had no influence on the inferred community composition, higher discharge reduced the number of Annelida and Ephemeroptera species detectable with eDNA. Most macroinvertebrate taxa showed the highest detection rates in spring, in particular merolimnic species with univoltine life cycles. Further, we detected an increase in proportion of shredders in winter and of parasites in summer. Our results show the usefulness of highly resolved eDNA metabarcoding time series data for ecological research and biodiversity monitoring in streams and rivers.