Quantitative information regarding reproduction is essential for conserving endangered animals; however, some conventional quantitative methods can be damaging to the target population and their habitats. In the present study, the reproductive migration of a threatened endemic fish, three-lips (Opsariichthys uncirostris uncirostris), was non-invasively monitored by quantitative PCR of species-specific environmental DNA (eDNA), the usefulness of which has been under-studied. Water sampling and from-shore visual inspection were performed weekly along a tributary of Lake Biwa (Japan), where adult fish seasonally migrate upstream to reproduce as well as at lake sites near the river mouth. Species-specific eDNA was collected at all locations at times when the fish were visually observed and at certain sites where the fish were not observed. Log-transformed individual counts from visual inspection were positively correlated with log-transformed eDNA concentration in the river sites, indicating that eDNA analysis can be a reliable quantitative tool for fish abundance in rivers. Furthermore, distance from the lake did not influence eDNA concentration, suggesting that eDNA transport by river flow had a negligible effect on eDNA quantification. eDNA concentration and individual counts both gradually increased from May–July, then both decreased in August. Importantly, eDNA analysis showed that the fish occupied more habitats in the peak reproductive season and stayed for longer time at any given site. An additional underwater survey confirmed unexpected eDNA detections as true positives. eDNA analysis has great potential to quantitatively monitor reproductive fish migrations under certain conditions.