Graphene membranes were prepared by laser ablation of polyimide membranes, and the graphene membrane with more oxygen-containing functional groups on surface was prepared by assisted oxygen plasma treatment. A scanning electron microscope was used to analyze the surface morphology of the membrane, and the prepared graphene had porous microstructures. The surface element content of graphene was analyzed by X-ray photoelectron spectroscopy. After oxygen plasma treatment, the oxygen content (atomic fraction) of graphene reached 15.6%. The contact angle measurement was used to characterize the underwater oleophobicity of graphene membranes. The contact angle of trichloromethane underwater was about 150°. This technology provides a feasible strategy for large-scale preparation of graphene membranes with the characteristic of underwater oleophobicity.