The effect of dynamic high pressure microfluidization on the microstructure of ovalbumin was studied by CD spectra, XRD spectra, ANS fluorescence probe emission spectra and UV absorption spectra. The results indicated that the changes in the microstructure were dependent on the pressure. CD spectra were used to examine the changes in the secondary structure of the ovalbumin treated by different pressures. When the pressure increased, the mutual transformation between α-helix, β-sheet, β-turn and the random coil was observed. The orderliness of the secondary structure was increased with increasing the pressure. XRD spectra analysis showed that the crystal structure content of the ovalbumin treated increased with increasing the pressure and the largest data was observed at 160 MPa, indicating that the orderliness of the secondary structure was increased. The results were similar to CD spectra analysis. The ANS fluorescence probe emission spectra analysis demonstrated that the dynamic high pressure microfluidization induced an increase in surface hydrophobicity following high pressure treatment, while the largest data was observed at 120 MPa. In addition, UV absorption spectra analysis indicated that dynamic high pressure microfluidization treatment also resulted in a decrease in the UV-absorption maximum wavelength with increasing the pressure, indicating that the aromatic amino acid was buried in the molecular interior and the three dimensional structure of ovalbumin was changed.