Chrome-tanned collagen fiber is the major solid waste of leather industry which is difficult to biological degradation and would caused serious pollution to the environment. However, the collagen fibers as a natural biological polymer materials, has unique properties of surface reactivity that the other polymer materials are not available. In this paper, the composite film of chrome-tanned collagen fiber- polyurethane (PU) were prepared is owing to H2O induced phase separation, and measured water vapor permeability, permeability, mechanical properties and microstructure. The results showed that the composite film of chrome-tanned collagen fiber-PU have continuous porous structure, which can improve the water vapor permeability and permeability of film, but reduce the mechanical properties of film. Thermal gravimetric analysis (TGA) showed that the thermal effect of collagen fiber and PU did not change significantly, so composite of collagen fiber and polyurethane by H2O induced phase separation belongs to physical process. The result demonstrates that this composite film has continuous porous structure, and collagen fibers with unique properties of surface reactions, so this material have potential applications in many fields.