Protein corona (PC) adsorbed on the surface of nanoparticles brings new research perspectives on the interaction between nanoparticles and fermentative microorganisms. Herein, the proteolysis of wheat PC adsorbed on a nano-Se surface using cell-free protease extract from S. cerevisiae was conducted. The proteolysis caused monotonic changes of ζ-potentials and surface hydrophobicity of PC. Notably, the innermost PC layer was difficult to be proteolyzed. Furthermore, when S. cerevisiae was stimulated by ultrasound + 0.1 mg/mL nano-Se@PC, the proportion of lethal and sublethal injured cells increased as a function of the proteolysis time of PC. The transcriptomics analysis revealed that 34 differentially expressed genes which varied monotonically were related to the plasma membrane, fatty acid metabolism, glycerolipid metabolism, etc. Significant declines in the membrane potential and proton motive force disruption of membrane were found with the prolonged proteolysis time; meanwhile, higher membrane permeability, membrane oxidative stress levels, membrane lipid fluidity, and micro-viscosity were triggered.