Large-scale biomonitoring is important to understand seasonal habitat use of amphidromous fish in river networks. This study used environmental DNA (eDNA) analysis to monitor spatiotemporal abundance patterns of an amphidromous fish (Ayu Plecoglossus altivelis altivelis) in a river network. We conducted eDNA samplings to monitor the seasonal habitat use of Ayu in the mainstem and tributaries of the Gonokawa River, Japan from summer to fall in 2022-2024. We used the quantitative PCR to estimate the eDNA concentrations (indicators of relative abundances) of Ayu in the study sites. In addition, we examined the relationships between the Ayu eDNA concentrations and a key environmental factor (tributary discharge) in the study sites. Our results indicated that the eDNA ratio of tributary to mainstem increased in the growing seasons and decreased in the spawning seasons, suggesting that Ayu could move between the mainstem and tributaries during the sampling period. Further, the eDNA concentrations were positively related to tributary discharge in the study sites. These results suggest that Ayu changed their habitats along their life history, and certain environmental factors could affect the seasonal habitat use of the target species in the riverine system.