Environmental DNA (eDNA) analysis provides an efficient and objective approach for monitoring and assessing ecological status; however, studies on the eDNA of aquatic insects, such as Ephemeroptera, Plecoptera, and Trichoptera (EPT), are limited despite its potential as a useful indicator of river health. Here, we investigated the community structures of aquatic insects using eDNA and evaluated the applicability of eDNA data for calculating assessment indices. Field surveys were conducted to sample river water for eDNA at six locations from upstream to downstream of two rivers in Japan in July and November 2016. Simultaneously, aquatic insects were collected using the traditional Surber net survey method. The communities of aquatic insects were revealed using eDNA by targeting the cytochrome oxidase subunit I gene in mitochondrial DNA via metabarcoding analyses. As a result, the eDNA revealed 63 families and 75 genera of aquatic insects, which was double than that detected by the Surber net survey (especially for families in Diptera and Hemiptera). The seasonal differences of communities were distinguished by both the eDNA and Surber net survey data. Furthermore, the total nitrogen concentration, a surrogate of organic pollution, showed positive correlations with biotic environmental assessment indices (i.e., EPT index and Chironomidae index) calculated using eDNA at the genus-level resolution but the indices calculated using the Surber net survey data. Our results demonstrated that eDNA analysis with higher taxonomic resolution can provide as a more sensitive environmental assessment index than the traditional method that requires biotic samples.