Abstract: The study was focused on the factors affecting AOT reverse micelle extraction of protein and mechanism, which could explain the reason for the extraction yield changes when extracting and separating protein and oil from AOT reverse micelle, and could further understand the mechanism of separation biomolecules using reverse micelle extraction. The results indicated that the molar ratio of water to surfactant (W0) increased with the increase of the viscosity of reverse micelle, and the capacity of extracting protein and oil from reverse micelle increased. According to Gibbs formula, it was concluded that reverse micelle of 0.08 g/mL AOT showed the higher interface activity and moisture (W0), which was in favor of the protein and oil extraction in reverse micelle. The structure of reverse micelle was investigated by fluorescence spectra and conductivity methods. The results showed that the reverse micellar system was quite stable at W0=11～14, AOT≥0.06 g/mL, and the AOT reverse micelle system seemed a combination type of a nonpercolation system and a percolation system, and the conductivity could reflect the changes of reverse micelle phase.