Objective To establish a method for the determination of isoflavone in soybean by ultra performance liquid chromatography tandem mass spectrometry with the contents of genistein， daidzein and glycitein as the indexes.Methods The samples were extracted by ethanol-water （3+1， V/V）， separated by Acquity UPLC^? BEH C18 column（3.0 mm ×150 mm， 1.7 μm） and then eluted by methanol and 0.1% formic acid solution （containing 5 mmol/L ammonium acetate） as mobile phase， detected by UPLC-MS/MS under the electrospray ionization （ESI^-） anion ion mode with multiple reaction monitoring mode. Results The linear relationship between genistein and daidzein was good in the range of 5.0-500 μg/L （r>0.995）， the detection limits were 2.7 and 4.0 mg/kg， respectively. The average recoveries at the addition levels of 300.0， 600.0， and 1 200 mg/kg were 89.4% to 102.2% and the relative standard deviations （n=6） were 1.5% to 4.6%. The linear relationship of glycitein was good in the range of 0.5-50.0 μg/L （r>0.995）， the detection limit was 0.6 mg/kg. The average recoveries at the addition levels of 30.0， 60.0， and 120.0 mg/kg were 85.7% to 104.0% and the relative standard deviations （n=6） were 1.3% to 2.8%.Conclusion The method is simple， sensitive and accurate， which is suitable for the determination of isoflavone in soybean.