Litopenaeus vannamei protein (LVP) is a high-quality protein. However, its functional properties do not fully meet the needs of food processing. In this study, LVP-xylose conjugates were prepared by conventional wet heat method (GLVP) and ball-milling-assisted wet heat method (GBLVP), respectively. The changes in structure and functional properties of the glycosylated LVP were explored. The findings revealed that ball-milling pretreatment increased the grafting degree to 35.21%. GBLVP had a sparser surface structure and lower particle size than GLVP. FTIR spectra showed that xylose was grafted onto LVP successfully and GBLVP had the lowest α-helix content. Compared with GLVP, GBLVP had a decrease in intrinsic fluorescence intensity and surface hydrophobicity, and an increase in UV absorption intensity. Moreover, GBLVP had higher foaming capacity, solubility and water-holding capacity, and lower allergenicity than GLVP. However, ball-milling pretreatment had a negative impact on the vitro digestibility and oil-holding capacity of GBLVP. In conclusion, ball-milling-assisted treatment of glycosylation could effectively improve the functional properties of LVP, benefiting the broader application of LVP in the food industry.