The invention relates to a method for preparing protein-based surfactant by opening protein disulfide linkage. The method is characterized by converting disulfide linkage into sulfonic group by oxidizing disulfide bond in protein of peroxide, and opening the folded coiling structure of the protein to represent the surface activity properties so as to prepare the protein-based surfactant which has the functions of emulsification, foaming, defoaming, washing, decontamination, soakage, and the like; by introducing polar group or nonpolar group into a protein side chain with opened disulfide bond, the surface activity properties of peptide fragments can be further improved or increased; moreover, by adopting treatments including acetylization, succinylation, acylation or phosphorylation, protein surface activity properties such as surface tension, emulsification, foaming, defoaming, washing, decontamination and soakage can be changed further. The method furthest prevents protein polar group and nonpolar group from being damaged, and maintains the structural integrity and property naturalness of protein molecules, thereby converting the protein into natural high-performance surfactant.