<p>Yeast extract is rich in nutrients and is a natural and safe food ingredient. The current process primarily focuses on extracting soluble components from yeast cells, however the utilization of yeast cell wall components remains underdeveloped, leading to the waste of valuable by-products. This study innovatively introduced microfluidization homogenization into the field of yeast extract production and established a high-pressure microfluidization homogenization (HPMH)-assisted yeast autolysis process. It was found that the free amino acid nitrogen content and cell damage index in the treated samples increased by approximately 24.3% and 33.8%, respectively, compared to the high-pressure homogenization (HPH) group; after 42&#xa0;h of autolysis, the protein, nucleic acid, extracellular protease activity, free amino acid nitrogen of the HPMH group samples increased by about 4.3%, 9.7%, 4.7%, and 10.8% respectively compared with the HPH group. The content of flavor amino acids in the yeast extract sample prepared using this method reached 49.78&#xa0;mg/g, accounting for 51.6% of the total amino acids, and the proportion of flavor amino acids was about 4.2% points higher than that of the enzymatic hydrolysis group. In brief, HPMH could effectively optimize the preparation process of yeast extract and its by-products.</p>