The invention discloses a composite double-layer film capable of regulating and controlling selective release of biomolecules through an electric field and a preparation method thereof. The film is continuously deposited through an electrochemical deposition method, the lower layer is a traditional doped polypyrrole film layer, and the upper layer is a polypyrrole composite layer which contains two kinds of biomolecules co-deposition; and the thickness and the composition of the upper layer and the lower layer can be regulated and controlled through the electrochemical deposition electric quantity and the deposition electric current and time. The film has good biocompatibility, in view of different changes of the biomolecules caused by the change of the pH near electrodes of the biomolecules and the large difference of the particle size and the electronegativity between the two kinds of biomolecules, selective release of the two kinds of in-situ biomolecules can be regulated and controlled through different voltages, and related growth factors needed by cells at different periods are provided for the cells at a fixed time and location; and the preparation process of the film is simple and feasible, the film can be widely applied to the fields of cell culture in vitro, tissue engineering, medical instrument surface coatings and the like.