Nitrogen (N) loading from domestic, agricultural and industrial sources can lead to excessive growth of macrophytes or phytoplankton in aquatic environment. Many studies have used stable isotope ratios to identify anthropogenic nitrogen in aquatic systems as a useful method for studying nitrogen cycle. In this study to evaluate the precision and accuracy of denitrification bacteria method (Pseudomonas chlororaphis ssp. Aureofaciens (ATCC® 13985)), three reference (IAEA-NO-3 (Potassium nitrate KNO3), USGS34 (Potassium nitrate KNO3), USGS35 (Sodium nitrate KNO3)) were analyzed 5 times repeatedly. Measured the δ15N-NO3 and δ18O-NO3 values of IAEA-NO-3, USGS 34 and USGS35 were δ15N: 4.7±0.1‰ δ18O: 25.6±0.5‰, δ15N: -1.8±0.1‰ δ18O: -27.8±0.4‰, and δ15N: 2.7±0.2‰ δ18O: 57.5±0.7‰, respectively, which are within recommended values of analytical uncertainties. Also, we investigated isotope values of potential nitrogen source (soil, synthetic fertilizer and organic-animal manures) and temporal patterns of δ15N-NO3 and δ18O-NO3 values in river samples during from May to December. δ15N-NO3 and δ18O-NO3 values are enriched in December suggesting that organic-animal manures should be one of the main N sources in those areas. The current study clarifies the reliability of denitrification bacteria method and the usefulness of stable isotopic techniques to trace the anthropogenic nitrogen source in freshwater ecosystem.