A method of high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) coupled with immunoaffinity purification column was developed for the determination of biotin in infant formula milk powder.The sample was dissolved with phosphate buffer and cleaned up through biotin immunoaffinity column.The analyte was separated on an Ultimate AQ-C18(2.1 mm×100 mm,3 μm) column using methanol-0.1% formic acid(30∶70,by volume) as mobile phase with injection volume of 5 μL,at a flow rate of 0.3 mL/min and a column temperature of 40 ℃.The analysis of biotin was performed by HPLC-MS/MS in multiple reaction monitoring(MRM) mode via positive electrospray ionization(ESI) source.The quantitative ion was m/z 227.2,and qualitative ions were m/z 227.2,96.9,with excitation voltages of 10,30 V.A good linear calibration was obtained for biotin in the range of 0.1-1.0 μg/mL，with a method detection limit of 20.3 μg/kg.The average recoveries at three spiked levels were in the range of 92.7%-98.5% with RSDs of 1.7%-1.9%.With the advantages of easy sample handling,high sensitivity,good reproducibility,short analysis time,the method could meet for the determination of biotin in infant formula milk powder.