Solvent resistance of graphene oxide (GO) makes it a promising material to construct membranes for precise separations with solvent-involved conditions. A thin GO laminate layer supported by a porous substrate displaying a thin film composite ( TFC) membrane configuration is commonly employed, and integration of the composite structure is necessary to acquire desirable performance. While the substrate effects on solvent transport in supported laminates were rarely involved, here we prepared integrated GO laminates using diamine crosslinking as a precondition and then investigated the effects of supporting substrates with different porous structure properties on TFC membrane performance. It was found that even if the laminates were integrated with similarly stable interlayer spacing in solvents, the substrate played a key role in forming GO laminates with desirable molecular rejection properties in solvents. The dye molecule rejection performance of the TFC GO membrane was significantly changed from 3% to 92% by optimizing the supporting substrate with limited surface porosity and pore size. Additionally, the altered stacking and channel formation of the GO nanosheets by the substrate exhibited solvent permeation following different transport models. This study may provide considerations in designing functional GO membranes for solvent resistant filtrations with an emphasis on substrate optimization.