Large defects have important impact on performance of separation membranes. In this paper, in order to reduce the large defects of the industrial ceramic supports, we prepared a nanohybrid separation membrane with a selective layer and a sub layer on the macroporous substrate with polyelectrolyte-coated nanoparticles. SEM, EDS and pore size analyses suggest that the although ZrO2 nanoparticles can migrate into the large defects to modify the membrane pores, the matching of pore-mouth size and diameter of NPs plays important role in the in situ modification of the substrates. The integrality of the as-prepared nanohybrid multilayer membrane was evaluated in the nanofiltration of dye solutions. The organic-inorganic composite membranes perform high flux and retention for separation. Membranes were prepared to study the separation performance on different substrates and with different building blocks (polyelectrolyte molecule weights and polyelectrolyte molecule structure). We found that the pore-mouth size of substrates and the structure of building blocks mainly affected the structure and the nanofiltration performance of the composite membranes. Such assembly allow us to in situ modify the large defects of inorganic substrates while preparing the selective layer. (C) 2017 Elsevier B.V. All rights reserved.