Summary Environmental DNA ( eDNA ) analysis for detecting the presence of aquatic and terrestrial organisms is an established method, and the eDNA concentration of a species can reflect its abundance/biomass at a site. However, attempts to estimate the abundance/biomass of aquatic species using eDNA concentrations in large stream and river ecosystems have received little attention. We determined the eDNA concentration and abundance/biomass of a stream fish, Plecoglossus altivelis , by conducting a snorkelling survey in the Saba River, Japan. Furthermore, we evaluated the relationship between eDNA concentrations and the estimated abundance/biomass of P. altivelis , and determined its spatial distribution within the river. Across the three seasons from spring to autumn, we found significant correlations between the eDNA concentration of P. altivelis and its abundance/biomass at study sites within the river. We detected the eDNA at the sites where we found only feeding traces on stones (where P. altivelis was not directly observed), but not at sites without feeding traces. Additionally, we tested the optimal number of qPCR replicates needed for the eDNA evaluation of P. altivelis abundance and biomass; only a small number of replicates was required when the eDNA concentration was high. Our findings suggest that eDNA analysis is a useful tool to estimate fish abundance/biomass as well as their spatial distribution in rivers.