Abstract: During aqueous processing of peanuts for simultaneous oil extraction and protein recovery, large amounts of emulsion could be formed and after enzymatic demulsification, substantial amounts of oil would be recovered while stubborn emulsions still remain. The destabilization of the stubborn emulsion is the key to improve the total free oil yield. Before its utilization and further destabilization, studying the characterization of the stubborn emulsion, especially its surface protein, which may play an essential role in emulsion stabilization, was necessary. The surface protein was extracted and its electrophoresis property, hydrophobicity, emulsifying activity, as well as emulsifying stability were studied. Confocal laser scanning microscopy (CLSM) was used to investigate its microstructure. It was found that, though the protein from the emulsion surface had similar subunits (60, 41, 38.5, 37.5, and 18 kDa) with that from aqueous phase, its hydrophobicity and emulsion activity was significantly higher. This could be attributed to the synergistic effect of temperature and pH during the alkaline extraction, which led to the unfolding of some large peanut protein molecules containing hydrophobic basic arachins. This, consequently, caused the exposure of more hydrophobic groups and enhanced the hydrophobic and emulsifying properties of the protein. Thus emulsion formation was promoted. After enzymatic treatment, the protein in the emulsion was hydrolyzed into short peptides and no subunits with molecular weight higher than 20 kDa had been detected in Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). However, in non-reducing PAGE, except for the conarachin band of 60 kDa, protein from the stubborn emulsion surface showed similar bands with that from the emulsion surface and aqueous phase. This indicated that the hydrolyzed protein could still gather on the stubborn emulsion surface and contributed to its stability. Due to the hydrolysis of protein molecules, the hydrophobic property and emulsifying activity of protein from stubborn emulsion was lower than that from an untreated emulsion surface. CLSM observation showed that stubborn emulsion had less oil droplets and that their size was lower, while the surface protein concentration (Γ) was higher, as compared with untreated emulsion. This explained the high stability of stubborn emulsion, though its surface protein has lower surface activity. To demulsify the stubborn emulsion, various treatments, including ultrasound, freeze-thaw, heating, extreme pH value, phase inversion, or ethanol addition were attempted. Free oil was obtained after centrifugation and total free oil yield was calculated thereafter. The microstructure of the stubborn emulsion after different treatments was also observed with CLSM. Results show that freeze-thaw and ethanol addition could remarkably aggregate the oil droplets in stubborn emulsion, especially after 50% ethanol addition, most oil droplets were combined and 90% of the oil in stubborn emulsion could be recovered. Under this condition, the total free oil yield could be increased to 93% from 88% in the overall process.