The invention discloses a method for preparing a biological protein two-dimensional nano-film by using lysozyme. According to the method, nano-particles which are generated by phase transformation reaction of lysozyme molecules and tris (2-carboxyethyl) phosphine hydrochloride are self-assembled on a gas-liquid interface through surface and interface induction to form a biological protein two-dimensional nano-film, or a base material is in contact with the surface of solution in a preparation process of the film, and the nano-particles which are generated by phase transformation of the lysozyme are directly self-assembled on a liquid-solid surface through surface and interface induction to form the two-dimensional nano-film, namely, the nano-particles grow on the surface of the base material in an in-situ manner. The preparation method is simple and environment-friendly; the thickness of the prepared biological protein two-dimensional nano-film is about 30-80 nanometers, surface roughness is small, transparency is high, adhesivity is good, and the method can be used for modifying surfaces of base materials.