Abstract: Hemp Protein Isolate (HPI) was used as raw material to modify HPI through ultrahigh pressure assisted enzymatic hydrolysis reaction. The SDS-PAGE electrophoresis characteristics, surface hydrophobicity, sulfhydryl content, FTIR and endogenous fluorescence of the hydrolysate of hemp protein isolate (HPIH) were determined under different pressures to investigate the structural changes of the HPI before and after modification. The results confirmed that ultra-high pressure (UHP) (0.1, 100, 200, 300 MPa) treatment had a certain auxiliary effect on HPI enzymolysis reaction, and with the increase of pressure, the degree of enzymolysis reaction increased gradually, and the molecular weight decreased gradually. After HPI modification, the hydrophobic groups were gradually exposed, and the surface hydrophobicity increased first and then decreased with the increase of pressure, the change difference was significant (P<0.05). The surface hydrophobicity reached the maximum at 200 MPa. After enzymolysis, the free sulfhydryl content of HPIH decreased significantly (P<0.05), while the surface sulfhydryl content increased first and then decreased with the increase of pressure. The determination of amino acid composition and content of protein before and after modification showed that the amino acid composition of HPI remained unchanged before and after modification, but the contents of various amino acids decreased to varying degrees. According to the fourier infrared spectroscopy, compared with HPI, the absorption peak intensity, peak shape and peak area of HPIH changed to different degrees, indicating that the secondary structure of protein was changed by the ultra-high pressure assisted enzymatic hydrolysis reaction. The endogenous fluorescence spectra showed that the fluorescence intensity of HPIH increased and the maximum emission wavelength was redshifted, indicating that the tertiary structure of HPI was changed by the enzymatic hydrolysis reaction. The results of antioxidant activity showed that appropriate pressure treatment could effectively improve the antioxidant capacity of enzymatic hydrolysis products. When the pressure was 200 MPa, the reducing power of HPIH of DPPH· and ABTS⁺· reached the highest. In conclusion, ultrahigh pressure assisted enzymatic hydrolysis modification can significantly change the secondary and tertiary structure of HPI, exposing hydrophobic groups and other active groups, thereby improving its antioxidant properties.