In this study, electron beam irradiation (EBI) technology was used to modify rice protein. The enzymatic hydrolysis efficiency of modified rice protein under different enzymes was studied with hydrolysis degree and polypeptide yield as evaluation indexes. It was found that EBI denaturation treatment could effectively improve the enzymatic hydrolysis efficiency of rice protein and increase the polypeptide yield, among which EBI assisted alkaline protease hydrolysis had the best effect, the rice protein hydrolysis degree increased by (19.02±0.37)%, and the polypeptide yield increased by (13.50±0.29)%. The SEM analysis showed that EBI denaturation treatment decreased the surface structure integrity of rice protein and increased the granulation degree. The content of α-helix decreased from (18.38±0.31)% to (4.46±0.43)%, indicating that the molecular flexibility of rice protein increased. The UV spectrum and endogenous fluorescence spectrum analysis showed that EBI denaturation technology expanded the spatial conformation of rice protein molecules, exposing more active groups embedded in the internal hydrophobic region. The structural changes of protein are beneficial to the enzymatic hydrolysis reaction.