L-Arginine (L-Arg) and lysine (L-Lys) with final concentrations at 1 g/L and 3 g/L were added to 20 g/L soy protein isolate (SPI) solution, respectively. The samples without the addition of L-Arg or L-Lys but with adjusting to the corresponding same pHs with of the added L-Ary or L-Lys were served as the control. These samples were then subjected to prepare oil-in-water (O/W) emulsions. The physical and chemical measurements and spectral scanning techniques were employed to detect the structure, physicochemical properties and emulsifying properties of SPI. Subsequently, the microstructure of the resulting emulsions was accordingly characterized. The results have shown that the addition of L-Arg and L-Lys increased the pHs of the SPI solutions and significantly improved the solubility (from 77.1% to max 91.3%), dramatically decreased the turbidity. Due to the protein folding of the hydrophobic group and resulting lowered protein hydrophobicity, L-Arg/L-Lys also effectively reduced the SPI particle size in the solution but also increased the charge, significantly reduced the size of emulsion droplets and promoted their uniformity. Consequently, the maximum 31.4% of the SPI emulsifying activity index and 78.9% emulsifying stability index of the L-Arg/L-Lys-treated samples were obtained. Comparatively, L-Arg was capable of modifying the SPI structure more effective than L-Lys to obtain enhanced emulsifying properties and the ameliorating effect was increased with the increasing concentrations of the two amino acids.