Microfluidization followed by density-based separation could effectively extract protein from broken rice with high protein recovery by disrupting protein-starch agglomerates, and follow-up enzyme treatments evidently removed residual starch of the protein-rich fraction to increase protein purity. Rice protein mainly composed of glutelin was non-destructively prepared by enzyme-assisted microfluidization. In comparison with AE-RP, EM-RP exhibited native structure, as demonstrated by lower solubility and the presence of an endothermic peak. Glutamic acid/glutamine, leucine, aromatic and charged amino acids were rich in the AE-RP. Moreover, EM-RP (mainly glutelin) showed higher digestibility than AE-RP due to the richness of glutelin (an easy-to-digest protein). These findings provide the food industry with a possibility for non-destructive and selective production of rice glutelin.