Liquid Egg yolk (LEY) tend to aggregate during pasteurization and lead to pipe blockage, which has become a thorny problem in food industry. In this paper, the mechanism of two common amino acid osmolytes (betaine and proline) delaying thermal aggregation of LEY was investigated. The result showed that addition of 3% betaine or proline improved thermal denaturation temperature of LEY in rheology and also reduced its viscoelasticity. The addition of amino acid shrinked particle size distribution of LEY. Betaine LEY (BLEY) had strong surface hydrophobicity while proline LEY (PLEY) had excellent solubility, both of which maintained its emulsifying property. Scanning electron microscopy (SEM) revealed that BLEY and PLEY had more porous surfaces, and the structure was more loose compared with LEY at the same temperature. The relaxation time T2 indicated that betaine and proline can alleviate conversion of free water to immobilized and bound water during heat treatment process, thereby reducing the degree of thermal aggregation and viscosity. Fourier transform infrared spectroscopy (FT-IR) demonstrated the tendency of α-helix transformed to β-sheet as heating progress. Compared with LEY, the structure of BLEY and PLEY was more ordered, suggesting that orderliness of structure was the foundation for delaying thermal aggregation. In conclusion, this study provided a new idea and theoretical basis for production of heat-resistant LEY.