Seeking highly efficient, rapid, universal, and low-cost demulsification materials to break up the crude/heavy oil-in-water emulsion and emulsified oily wastewater at ambient conditions has been the goal of the petroleum industry. In this work, an amphiphilic material, graphene oxide (GO) nanosheets, was introduced as a versatile demulsifier to break up the oil-in-water emulsion at room temperature. It was encouraging to find that the small oil droplets in the emulsion quickly coalesced to form the oil phase and separated with the water within a few minutes. The demulsification tests indicated that the residual oil in separated water samples was as low as ∼30 mg/L, corresponding to a demulsification efficiency over 99.9% at an optimum GO dosage. More importantly, GO not only is useful for ordinary crude oil emulsion but also can be used to break up the extra heavy oil emulsion. The effect of the emulsion pH on the demulsification was also investigated. It was interesting to find that the distribution of GO either in oil or in water phase after demulsification was dependent on the pH value of the solution, which was attributed to the pH-dependent amphiphilicity of GO. The prominent demulsification ability of GO was attributed to the strong adsorption between the GO nanosheets and molecules of asphaltenes/resins driven by π–π interactions and/or n−π interactions. The findings in this work indicate that the GO nanosheets are a simple, highly efficient, and universal demulsifier to separate the oil from the crude/heavy oil-in-water emulsions at ambient conditions, which shows a good application prospect in the oil industry.