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Abstract Long‐term biodiversity monitoring is necessary for conservation and management. In such circumstances, environmental DNA (eDNA) surveys can enable easy and effective biomonitoring of aquatic insects. However, previous studies of aquatic insects based on the mtDNA COI region have revealed incomplete taxonomic coverage, and frequent amplification of nontarget taxa (e.g., algae and diatoms). Additionally, it has been indicated that there are few reference sequences registered in databases for metabarcoding analysis of insects. Therefore, we developed a new primer set, MtInsects‐16S, for eDNA analyses of insects based on the mtDNA 16S rRNA region in a previous study. To address the insufficient database records of insect DNA sequences, we also constructed a comprehensive reference database of aquatic insects that occur in Kanagawa Prefecture, Japan. We conducted eDNA analyses at six sites in the two river systems (the Sagami‐gawa River and Sakawa‐gawa River systems), with three replicates per site, based on both the mtDNA COI and the 16S rRNA regions. These results were compared physical capture surveys at the same sites to examine the detection capability of eDNA for Insecta with using a well‐established database. Among the list of species which were collected by physical capture surveys, 74.9% were detected by MtInsects‐16S, whereas 40.1% were detected by the primer set of the mtDNA COI region, and also 80.0% were detected by the both primer sets. This study demonstrated that the application of eDNA analyses using the MtInsects‐16S primer set can be conducted with accuracy and reliability, provided that the reference DNA database is improved. The MtInsects‐16S region can be considered indispensable in eDNA analysis for aquatic insects.