The purpose of this study was to detect the effects of lipids on the properties of emulsified myofibrillar protein (MP) interfacial layer by Raman spectroscopy combined with the protein adsorption characteristics. Results revealed that compared with butter and lard groups, the α-helix content in the soybean oil emulsifying layer was decreased (P < 0.05), while β-sheets and β-turns were increased (P < 0.05). The tryptophan bands and the disulphide bonds were higher in the soybean oil emulsifying layer, which implied a secondary structure, covalent bonding and hydrophobic interactions between the different lipids and MP have distinctive features. Moreover, the absolute value of the zeta potential in the soybean oil emulsifying layer was the highest, and the unit interface membrane protein surface loading (Γ S ) was higher, indicating that the soybean oil could form a more stable emulsifying layer with MP.