Living fish excrete ammonium (NH4+), and importance of excretion has been recognized. Nitrogen isotope ratio (δ15N) of NH4+ may be useful for quantification of fish-derived NH4+. We characterized δ15N of fish-excreted NH4+ and investigated whether the effect of fish excretion can be assessed from the δ15N of NH4+ in river water. The study was carried out in August 2020 and February, April, and August 2021 in the Chinai River. Large migration of iteroparous three-lips (Opsariichthys uncirostris) during summer, and increase of NH4+ concentration in the river water due to their excretion were observed. Three-lips were incubated, and δ15N of excreted NH4+ was measured. River water was sampled from several sites with different fish densities, and concentrations and δ15N of NH4+ were measured. δ15N of excreted NH4+ (+21.1‰) was higher than muscle (+16.7‰). No increase in concentrations and δ15N of NH4+ in river water during non-migratory periods were observed. During migratory periods, concentrations and δ15N in lower sites with high fish density were increase. The δ15N of endmember (+21‰) were consistent with the excretion, suggesting that the increase in concentration can be explained by excretion. Therefore, δ15N of NH4+ may be possible to evaluate fish-excretion in river water.