Objective To establish a method for the determination of selenium species in Se-enriched garlic with high performance liquid chromatography-inductively coupled plasma tandem mass spectrometry (HPLC-ICP-MS/MS). MethodsZORBAX SB-Aq column was used as analytical column; 10 mmol/L citric acid (ammonium citrate), 5 mmol/L sodium hexane sulfonate and 2% methanol were used as mobile phase for gradient elution. Baseline separation of selenate (SeO2-₄), selenite (SeO2-₃), selenocysteine (SeCys2), methylselenocysteine (MeSeCys), selenomethionine (SeMet) and Selenoethionine (SeEt) were completed in 10 min. Constant temperature oscillating assisted enzyme extraction was used to extract selenium species in garlic samples. Results The detection limits for SeO2-₄, SeO2-₃, SeCys2, MeSeCys, SeMet and SeEt were 0.40,0.40,0.44,0.47,0.35 and 0.45 μg/kg, respectively. The linear correlation coefficients (r) were all above 0.999 in the range of 0-100.0 μg/L. Except SeCys2, the recoveries were between 79.0%-104.0% and the relatives standard deviations were within 2.2%-5.0%. Conclusion The result showed that the method had good stability with high sensitivity and accuracy, and was suitable for determination of five selenium species in garlic.