Abstract: 0.1 g of defatted selenium-enriched soybean sample was taken into a centrifuge tube, and the sample was ultrasonicated with 5 mL of 130 mmol·L^-1 tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution at pH 7.5 for 30 min, then enzymatically hydrolyzed with 15 mg of pronase for 5 h at 37 ℃, and centrifuged to take the supernatant, which was passed through 0.22 μm nylon organic filter membrane. Selenocysteine (SeCys), selenomethionine (SeMet), and methylselenocysteine (MeSeCys) in the filtrate were separated on Agela MP-C₁₈ column, and eluted isocratically with 40 mmol·L^-1 ammonium hydrogen phosphate solution (pH 7.0) containing 2%(volume fraction) methanol and 0.5 mmol·L^-1 tetrabutylammonium bromide. The three selenoamino acids were determined by hydride generation atomic fluorescence spectrometry. As shown by the results, the baseline separation of the three selenoamino acids could be achieved within 7 min, and the linear ranges of the standard curves were all 5-100 μg·L^-1, with detection limits of 0.086, 0.075, and 0.047 mg·kg^-1 respectively. The recoveries of the three selenoamino acids according to the standard addition method were in the range of 85.5%-103%, and RSDs of the determined values(n=7) were not greater than 3.0%. This method was applied to analysis of the selenoamino acids in selenium-enriched soybeans, and the selenium fugitive form in soybeans was mostly SeMet, which accounted for 85.5%-94.5% of the total selenium.