The structure characteristic of soybean selenoprotein and soy protein isolate (SPI) were investigated with fluorescence, ultraviolet and Fourier transform infrared (FTIR) spectrum. The unfolding process of two proteins was analyzed with fluorescence phase diagram method. The stability of emulsion properties and the influence of concentration, temperature and pH on the conformation of soy selenoproteins were also determined. The results indicated that the covalent disulfide bond of soybean selenoprotein molecules was damaged; the hydrogen bonding become weak; the hydrophobic interactions were enhanced and the protein chain molecules were extended. Soybean selenoprotein displayed only “folding” and “loose” state in solution, which illustrated soybean selenoprotein more tend to hydrolysis when compared with soybean protein. With temperature increasing, the fluorescence quenching effect occurred and the hydrophobicity of soy selenoproteins was also gradually increased, which reflected the protein molecules tends to be folded. In the range of pH 2.8～8.0, the Trp residue of soybean selenoprotein was mainly distributed in the polarity of the external environment and presented different conformational change on both sides of the isoelectric point under different pH value. In acidic environment, the soybean selenoprotein was easy to appear conformational transition from loose to fold. But it was conducive for soybean selenoprotein to existence in loose structure in alkaline conditions. In addition, the emulsifying properties of soybean selenoprotein were analyzed based on UV spectral data. Results showed that lower temperature helps to enhancement the emulsification but unfavorable the stability of the soybean selenoprotein.