In this article， the structure and properties of the vitamin D3 (VD3) and soy protein isolate (SPI) complex were characterized by surface hydrophobicity， particle size， ζ-potential， turbidity， fluorescence spectroscopy， ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy (FTIR). The results showed that， the surface hydrophobicity decreased significantly (P<0.05) with the addition of VD3， and the content of VD3 was inversely proportional to the surface hydrophobicity of SPI. The addition of VD3 significantly reduced the particle size of SPI， and increased the absolute value of the ζ-potential of SPI. This indicate the particle size distribution of the solution droplets is more uniform， and the stability of the solution is stronger. With the increase of VD3 content， the turbidity of the complex increased slightly. Fluorescence spectrum and ultraviolet-visible spectrum analysis found that， compared with the SPI control， the maximum emission wavelength and maximum absorption wavelength of the complex were red-shifted 3.6 nm and 8 nm， respectively. This indicate that VD3 changes the spatial structure of SPI， increasing the polarity of the microenvironment of aromatic amino acid residues. Fourier infrared spectroscopy shows VD3 causes the secondary structure of SPI to change， the content of α-helix and β-sheet decreases， and the content of β-turn and random coils increases. The result of the research provided the basis for the technology of nutritional enhancement of VD3 and the widening of the application of soy protein.