Abstract: Oxygen stable isotope ratio (δ¹⁸O) of water is currently applied for wine authenticity assessment. The method for the determination of δ¹⁸O in water from wine samples was reported by GasBenchⅡ-IRMS using on-line equilibration technique. The effects of sample volume, equilibration time and ethanol content on the determination of δ¹⁸O in water were discussed. Results show that the method is stable with a standard deviation (1δ) lower than 0.1‰， when sample volumes range from 200 μL to 1 000 μL with 24—48 h equilibration times. This method has an advantage of easy and rapid operating with lowering sample consumption, and especially can be used continuously for a large number of instances in batch. Results also show that there is a positive liner relationship between tap water content and δ¹⁸O of water in spiked wine, which will be a suitable probe for detecting the illegal addition of water.