Effects of dynamic high pressure microfluidization (DHPM) pretreatment (0, 40, 80 and 120 MPa) combined with dextran glycation on thermal stability and structure of β-lactoglobulin (β-Lg) were investigated. The results showed that the peak temperature of β-Lg is 73.48 ℃, which decreased first and then increased after pretreated by DHPM at 40, 80 and 120 MPa. However, the thermal stability was improved after DHPM pre-treatment combined with glycation. Physiochemical analysis revealed that glycated β-Lg pretreated with DHPM at 80 MPa gave the lowest free amino (2.20 mg/mL) and the highest browning degree (A294=1.092，A420=0.620), suggesting that DHPM pre-treatment can promote glycation of β-Lg and dextran, and 80 MPa is the best pressure. Structural analysis indicated that DHPM pre-treatment can obviously improve the surface hydrophobicity and free sulfhydryl groups content, reduce the intrinsic fluorescence intensity, and change the secondary structure of β-Lg. After DHPM pre-treatment and glycation, the surface hydrophobicity of β-Lg was significantly reduced, but still higher than that of natural β-Lg, the content of free sulfhydryl groups increased first and then decreased with pre-treatment pressure, β-Lg content in 80 MPa pre-treated group was much higher than that of natural β-Lg. The intrinsic fluorescence intensity showed an adverse change trend with free sulfhydryl groups, but it still much lower than that of natural β-Lg. Therefore, β-Lg pre-treated with 80 MPa has the highest thermal stability and glycation degree, and the higher the glycation degree of β-Lg resulted, the better thermal stability it will be.