A gas chromatography-triple quadrupole mass spectrometric method was developed and validated for the determination of methyl selenocysteine in middle-aged milk powder.The analytes were extracted with trichloroacetic acid and acetonitrile,and derived with ethyl chloroformate in ethanol-pyridine.Methyl selenocysteine was separated on an HP-5MS micro-column,and quantified under multiple reaction monitoring mode.The calibration curve was linear in the range of 0.005 – 1.0 mg/L,with detection limit of 0.01 mg/kg and quantitation limit of 0.05 mg/kg.The average recoveries of methyl selenocysteine from middle-aged milk matrixes at the spiked level of 0.05,0.2,0.4 mg/kg ranged from 79% to 86% with relative standard deviations of 2.8%-4.0%.The method could meet the requirement for determination of methyl selenocysteine in middle-aged milk powder.