This paper studies the improvement of freeze–thaw stability of emulsions by incorporating glucosamine into soy protein isolate (SPI) by using transglutaminase. The occurrence of enzymatic glycosylated SPI reaction is confirmed by SDS-PAGE. Through the measurement of creaming index and oiling off, we find that both soy protein isolate-enzymatic without glucosamine (SPI-E) and glycosylated SPI by transglutaminase (SPI-G) can effectively improve the freeze–thaw stability of emulsions. The analysis of particle size, degree of flocculation and coalescence indicates that macromolecular aggregates were formed because transglutaminase enzyme promoted glycosylation and also promoted the cross-linking of protein and protein or the internal cross-linking of protein. Therefore, the particle size of the SPI-G emulsion before freezing and thawing is larger than that of the SPI emulsion. After freeze–thaw cycles, it can be seen that although the particle size of SPI-G is increasing, it is significantly smaller than that of SPI emulsion after freeze–thaw. According to the optical microscope, it can be seen that the SPI emulsion has obvious aggregation of oil droplets after freeze–thawing, and SPI-G effectively improves oil droplet aggregation.