The invention provides a preparation method and application of a graphene oxide skeleton material self-assembly membrane. The preparation method comprises the steps of firstly, providing a carrier, and carrying out charge modification on the surface of the carrier by using an amino-containing group; then, enabling a group containing double active terminals to have a thermal reaction with a graphene oxide solvent so as to prepare a graphene oxide skeleton structure solution with controllable interlayer spacing; finally, adopting a chemical deposition self-assembly method to enable the graphene oxide skeleton structure solution to form a dense self-assembly membrane on the surface of the carrier. The method improves the combining capacity of graphene oxide and the carrier by carrying out charge modification on the carrier. The graphene oxide skeleton material self-assembly membrane prepared by the method has excellent gas, liquid and ion selectivity, has higher compactness and mechanical strength, and has adjustable screening pore path and membrane thickness.