Abstract: In order to verify the applicability, reproducibility and accuracy of bacterial denitrification method for the determination of N and O isotope composition of nitrate in water, denitrifying bacteria were used to convert nitrate to N₂O in sea water, lake water and tap water samples on 28 July, 19 and 26 August of 2019 . The results show that the slope of the nitrogen isotope calibration curve of nitrate obtained from the 3 batches of experiments in different time periods was close to its theoretical value of 1, and the correlation coefficient was higher than 0.999. It indicates that the nitrogen isotope fractionation effect caused by denitrifying bacteria was relatively low when transfering from nitrate to N₂O in the samples. The nitrogen isotope values of nitrate obtained from the same samples in the 3 batches were basically the same, indicating that the determination of nitrogen isotopes of nitrate by bacterial denitrification method had good reproducibility and accuracy in a long time period. The slopes of the oxygen isotope calibration curves of the 3 batches were stable between 0.61 and 0.63. The correlation coefficients were higher than 0.99. And the standard deviation of oxygen isotope ratio of nitrate in 3 types of water samples in each batch ranged from 0.18‰ to 0.69‰. The correction results demonstrate that the oxygen isotope composition of nitrate in the samples can be accurately reflected by the respective oxygen isotope calibration curves. The range of oxygen isotope values of the same sample was 1.33‰ to 16.38‰. It was very different from each other among the 3 batches due to the reason probably caused by the oxygen isotope exchange between nitrate and water during the storage.