Graphene oxide (GO) has been considered as a promising material to develop advanced nanofiltration membranes benefiting from its extraordinary physicochemical properties. GO membranes for practical application need porous substrates to provide sufficient mechanical strength. Compared to extensive studies on the manipulation of GO selective layer, the influence of substrates on the performance of GO membranes has been received much less attention. Actually, significant differences in physical and chemical properties of the substrates should lead to distinct assembly structure of resulting GO membranes with uneven performance. Therefore, the effect of substrate on GO membranes formation and separation are worth study and optimization. Herein, we employed typical inorganic ceramic tube and polymeric polyacrylonitrile (PAN) and polycarbonate (PC) substrates to support GO membranes and studied the effects of their surface morphologies and roughness, surface chemical composition, as well as bulk pore structure on the formation and nanofiltration performance of resulting GO membranes. Substrates properties were revealed to have remarkable impacts on the adhesion and transport property of GO membranes. We found that the surface morphological and chemical structure of substrates induced GO assembly and determined GO adhesion, and the bulk pore structure of substrates dominated the whole transport resistance of GO membrane. Especially, the PAN substrate possessing abundant oxidized functional groups after simple hydrolysis, contributed to a robust interfacial adhesion with GO selective layer and enabled GO membrane to withstand harsh stability measurements including cross-flow, high feed pressure and long-period continuous operation. Besides, the smooth surface morphology along with the bulk highly porous structure of PAN substrate offered a favorable platform for GO assembly, resulting in competitive nanofiltration performance with water permeance of 15.5 Lm(-2) h(-1) bar(-1) and dye rejection of 99.5%. Overall, this work gives new insights of design and fabrication of durable GO membranes for practical applications.