In recent years, aquatic biodiversity surveys using environmental DNA (eDNA) have been conducted, but the behavior of eDNA remains largely unknown. In this study, we investigated the temporal variation of eDNA concentration of the Plecoglossus altivelis and Stenopsyche marmorata. As a result, the DNA of S. marmorata was detected from night to morning, while the DNA of P. altivelis was detected throughout the day. This may be related to the migratory and spawning habits of S. marmorata from evenings to mornings. Moreover, the detection frequency of S. marmorata increased when using a filter with a smaller pore diameter (0.20 µm) compared to a filter with a larger pore diameter (0.65 µm) typically used for eDNA analysis.