Abstract: Selenium is an essential trace element for human body. Insufficient intake of selenium can lead to Keshan disease, Kashin-Beck disease, cardiovascular and other diseases, while excessive intake can lead to selenium poisoning. There are diverse selenium species in the world. Different species of selenium possess different biological activities, migration and transformation rules, and have different effects on nutrition and health. The selenium level of serum is one of the most widely used sample for biological testing of selenium level. In order to study the species and level of selenium in human serum, the chromatographic conditions were optimized. Human serum and without macromolecular protein human serum were selected as samples for ultrasonic enzymatic hydrolysis pretreatment. ZORBAX SB-Aq C18 reversed-phase chromatography column was analysis column, 10 mmol/L citric acid and 5 mmol/L sodium hexane sulfonate (containing 1% methanol, pH 4.0) were mobile phase with isocratic elution. Selenate (Se(Ⅵ)), selenite (Se(Ⅳ)), selenocysteine (SeCys2), methylselenocysteine (MeSeCys) and selenomethionine (SeMet) in human serum samples were determined by high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The results indicated that five selenium species could be separated within 10 min. The linear of five selenium species was good in the range of 0.0-100.0 μg/L, and the correlation coefficients (r) were more than 0.999. The limits of detection were 0.05-0.2 μg/L. The reproducibility of the method was determined by precision. The relative standard deviations (RSDs) were all less than 5%. The standard recovery experiment was carried out by adding three concentration levels and five kinds of selenium form mixed standard solutions. The recoveries of Se(Ⅳ) were 35.1%-39.3% and 87.9%-90.0% for human serum and without macromolecule protein human serum, respectively. The recovery of SeCys2 was 57.7%-72.5%, while the recoveries of Se(Ⅵ), MeSeCys and SeMet were 80%-120%. Analysis of serum in 30 subjects indicated that the main species of selenium was SeCys2 with the concentration of 16.2-29.3 μg/L, followed by SeMet with the concentration of 6.2-16.3 μg/L. Additionally, a small amount of inorganic selenium and unknown selenium species were also determined in serum. Low recovery rate of Se(Ⅳ) in human serum may be due to combination between Se(Ⅳ) and macromolecular protein, which needs to be further studied.