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The accuracy of environmental DNA (eDNA) methods developed as innovative biological monitoring technologies can be improved by considering the ecological characteristics of a target species. In this study, we performed an aquarium experiment and field surveys to establish an effective eDNA method for an endangered Japanese bagrid catfish (
Pseudobagrus ichikawai
) with nocturnal habits. First, we developed a species-specific eDNA method using quantitative real-time polymerase chain reaction (qPCR) for
P. ichikawai
. In the aquarium experiment, we demonstrated that the addition of benzalkonium chloride (BAC), a reagent that inhibits eDNA degradation, was effective for water sample storage. In the field, eDNA has consistently been detected at sites where
P. ichikawai
has been confirmed. There was no difference in eDNA concentration between the water samples taken at different sampling times or between those taken from pools and riffles in the rivers. This result provides evidence that researchers can collect water samples during the daytime, which would reduce the risk of water accidents (e.g., drowning or submerged in darkness) associated with field eDNA surveys of nocturnal species, such as
P. ichikawai
. Furthermore, we propose that researchers perform on-site surveys for collecting water samples without considering the microhabitat preferences of the target species (e.g., riffle/pool). We recommend that water samples be treated with BAC immediately after collection in the field and then stored in low-temperature conditions. The cooling procedure with BAC outlined here can be advantageous in collecting samples from many sites in a short time, because it only requires the collection of water in the field.