Determination of hydrogen and oxygen isotope ratios in water samples by GasBench ⅡIRMS using aqua equilibration technique was reported in this paper. Effects of sample volume, aqua equilibration time and signal intensity of reference gas on the determination of isotope ratios of hydrogen and oxygen were discussed. CO2—H2O online equilibration method for determination of oxygen isotope ratios and hydrophobic platinum catalytic H2—H2O online equilibration method for determination of hydrogen isotope ratio by GasBench ⅡIRMS were established. The methods have been applied to hydrogen and oxygen isotope ratio determination in standard water samples. The results are in agreement with certified values with precision of 1‰ (1σ) for both hydrogen and oxygen isotope ratio determination. The methods provide the advantages of good reliability, high efficiency and less sample loss.