A detection method for five selenium species in Se-enriched rice was established using HPLC-ICP MS/MS. The ion pair concentration of mobile phase, elution salt concentration, pH value were optimized by using a reverse C18 chromatographic column, and the final mobile phase composition was determined to consist of 15 mmol/L ammonium acetate solution, pH5.5and 0.2 mmol/L TBAH, the baseline separation of SeMeCys,SeCys, SeMet, Se(VI) and Se(IV) was obtained. Selenium species in rice were extracted by enzyme extraction method. The effects of enzyme selection, enzyme dosage and extraction time on extraction efficiency were investigated. The optimal ratio of sample to protein XIV enzyme content was determined to be 20∶1, and the optimal enzymatic hydrolysis time was 20 h. The method has high separation efficiency, good linearity, good precision, and high sensitivity, and the recovery was 77.1%~107.3%. The limits of detection and the limits of quantitation of SeMeCys (methylselenocysteine), SeCys (selenocystine), and SeMet (selenomethionine) were 0.001 5 mg/kg and 0.005 mg/kg, respectively, while the limits of detection and the limits of quantitation of Se (VI) and Se (IV) were 0.002 5 mg/kg and 0.009 mg/kg. The method provide technical support for objective and comprehensive evaluation of the quality of Se-enriched rice and subsequent assessment of dietary exposure to Se-enriched rice.