Abstract: Sample of selenium-enriched yeast was extracted with Tris-HCl buffer solution of pH 7.5 by ultrasonic smashing for 30 min. Protein hydrolytic enzyme Pronase E was added to the extract, which was stirred at 37 ℃ for 18 h and separated by high speed centrifugation. The supernatant was taken and filtrated with Millipore Corp super-filtering membrane. Waters Symmetry Shield RP18 chromatographic column was used for separation of inorganic selenium (Ⅵ and Ⅳ) and organic selenium compounds of Selenocysteine, Selenourea and Seleno-methionine. Mixtures of methanol and heptafluoro-butanoic acid mixed in different ratios were used as mobile phase in gradient elution for the 5 species of selenium. The eluates were used for ICP-MS determination. Linear relationships of the 5 species of selenium were kept in the same range within 100 μg·L-1. The proposed method was applied to the analysis of sample of selenium-enriched yeast. It was found that: the amount of inorganic selenium was only amounting to 1.63% of the total selenium. The major species of selenium existed in yeast was selenomethionine, amounting to 65%, which was easily absorbed and utilized by the human body.