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 local and regional environmental factors in the mainstem and tributaries. Our results indicated the spatial position was significantly related to the mainstem Ayu eDNA concentrations, suggesting that fish upstream dispersal was the primary factor that determined the Ayu spatial patterns in the mainstem. In contrast, local environmental factors such as habitat size were correlated with the Ayu tributary eDNA concentrations, suggesting the local process could be important for the Ayu populations in the tributaries. These results suggest that the effects of local and regional processes on Ayu populations could spatially vary in the river network.