The vitamin B12 is an essential vitamin and participates in the biochemical reaction of the human body, including folic acid conversion and methionine synthesis. However, it is easy to lose its nutrition and function in the food processing process and storage such as light, heating and pH value. In order to improve the stability of the vitamin B12 and the vitamin B12 nutrition fortified food, the interaction between vitamin B12 and soybean protein isolate and the effect of vitamin B12 on protein structure were studied by using spectroscopic techniques (fluorescence spectrum, ultraviolet spectrum, infrared spectrum and circular dichroism spectrum). The result of fluorescence spectrum showed that the fluorescence intensity of soybean protein isolate was decreased with the increase of vitamin B12 concentration. The quenching mode of vitamin B12 on soybean protein isolate was static quenching by calculating the Stern-Volmer equation. Soybean protein isolate and vitamin B12 were bound by Van der Waals force and hydrogen bonding and binding site was one. Synchronous fluorescence spectrum indicated that the binding site of vitamin B12 and soybean protein isolate was located near tryptophan. Ultraviolet absorption spectrum presented that vitamin B12 enhanced hydrophobicity of the microenvironment near the tryptophan residue and changed the structure of protein molecules. The results of infrared spectrum and circular dichroism spectrum showed that the secondary structure of soybean protein isolate was changed with the addition of vitamin B12, which represented that the contents of α-helix and β-rotation were increased, and the contents of β-fold and irregular curl were decreased. 