Egg yolk sphere is a kind of natural colloidal histological structure formed by the co-assembly of a large number of proteins and lipids, which has the dual functions of hydrophilic and lipophilic for application in emulsion. In this study, egg yolk sphere microgels (EYSMs) were prepared by high pressure homogenization (HPH) technique at various pressures (0–90 MPa) and used as Pickering emulsifiers for emulsification to obtain oil-in-water emulsions with an oil-water ratio of 11:9. The microscopic morphology, structure, and physicochemical properties of EYSMs were analyzed. The results showed that EYSMs obtained at 60 MPa (60-EYSMs) exhibited superior emulsifying properties with an emulsifying activity index (EAI) of 2.8 m2/g and an emulsifying stability index (ESI) of 356.6 min, resulting in enhanced storage stability of the emulsion for 30 d. The HPH treatment decreased the lipid content in EYSMs while creating a more homogeneous protein-lipid arrangement, which provided EYSMs with balanced hydrophilic and hydrophobic sites to improve emulsifying properties. Furthermore, the free sulfhydryl groups and hydrophobic moieties exposed by HPH promoted -S-S- formation on the EYSMs surface, promoting the increase in surface hydrophobicity and emulsifying ability. The confocal laser scanning microscopy (CLSM) and cryogenic scanning electron microscopy (cryo-SEM) revealed that the EYSMs adsorbed at oil-water interface and formed an intercross-linked spatial three-dimensional network structure, which worked as a physical barrier to the stable emulsion formation. This study introduces a novel research concept and delves into the emulsification mechanism of protein-lipid composite particles, offering a fresh insight for the development and utilization of emulsifiers.