Milk fat globule membrane (MFGM) proteins have several biological functions and maintain the fat globule structure. However, the major MFGM protein compositions in simulated human milk emulsions are different from those in human milk due to the composition loss in the isolation process of MFGM materials. To overcome this limitation, we developed a novel strategy, namely, the solution enriched with MFGM was homogenized with cream separated from the milk rich in large-sized fat globules. The results of physicochemical properties and the interfacial protein coverage of the emulsions showed that the emulsions prepared by the new method had a smaller particle size, higher stability, and more interfacial protein coverage when the ratio of fat to protein was 1:3. In addition, proteome differences in interfacial proteins between the new emulsions and simulated infant formula emulsions were investigated, and the results revealed that the interface of the emulsions prepared by the new method contained all major MFGM proteins and unique GO annotations and KEGG pathways. However, only four MFGM proteins (XO, ADPH, PAS 6/7) were quantified at the interface of the emulsions prepared by the common method. Furthermore, the protein number and the total relative abundance of major MFGM proteins were approximately 2-fold and 475-fold higher at the interface of the emulsions prepared by the new method compared to the common method. Overall, the study modulated the interfacial protein composition of fat globules by screening the sources of lipid and homogenization methods and revealed its potential effect on processing stability and biological properties.