The invention discloses a preparation method of a self-assembled interlayer spacing adjustable rGO composite film, and belongs to the field of film separation. The preparation method comprises the following steps: mixing a certain concentration of an oxidized graphene solution and a certain concentration of an intercalation material at a certain temperature, stirring to configure a casting film solution, and then assembling the film in a fractionated assembling manner on a dopamine pretreated porous base film with rich amido through a vacuum-assisted self-assembling technology. The preparationmethod is simple in preparation process, short in film formation time and few in used materials of the film material, is applied to nanofiltration research, and solves the problem that the oxidized graphene composite film is small in flux and low in reject rate in the evaluation process of nanofiltration performance; the surface of the composite film assembled in a fractionated manner is more uniform and tight; and the preparation method can be widely applied to the field of water treatment.