Membranes synthesized by stacking two-dimensional (2D) graphene oxide (GO) hold great promises for improved permeability and separation capability in organic solvents. However, the separation capability of a layer-stacked GO membrane in organic solvents can be significantly affected by its swelling and interlayer spacing, which has not yet been systematically characterized. In this study, the interlayer spacing of a layer-stacked GO membrane in different organic solvents was experimentally characterized by liquid-phase ellipsometry. To understand the swelling mechanism, the solubility parameters of GO were experimentally determined and used to mathematically predict the Hansen solubility distance (Ra) between GO and solvents, which is found to be a good predictor for GO swelling and the interlayer spacing. Solvents with low solubility distance (e.g., dimethylformamide, n-methyl-2-pyrrolidone) tend to cause significant GO swelling, resulting in an interlayer spacing of up to 2.7 nm. Solvents with high solubility distance (above 10) such as ethanol, acetone, hexane and toluene only cause minor swelling and are thus able to maintain an interlayer spacing of around 1 nm. Correspondingly, GO membranes in solvents with high solubility distance exhibit better separation performance, for example, more than 90% rejection of small organic dye molecules (e.g., rhodamine B and methylene blue) in ethanol and acetone. Additionally, solvents with higher solubility distance results in a higher slip velocity in GO channels and thus higher solvent flux through the GO membrane.