Abstract A molecular layer-by-layer (mLbL) technique was recently developed to fabricate polyamide (PA) thin film composite (TFC) membranes for water purification. In this study, the interlayer structure between the selective and support layers of the mLbL-assembled TFC membrane was tailored to achieve high performance applicable to seawater desalination. Introducing interlayers on porous supports prior to mLbL deposition allowed the effective PA growth by preventing monomer deposition within the support pores. The PA layers were grown via mLbL on supports coated by a series of interlayers: poly(piperazine-amide), cross-linked poly(ethyleneimine) (PEI) and a polyelectrolyte bilayer of PEI and poly(acrylic acid) (PAA) (PEI/PAA). The density and distribution of surface carboxyl groups of the interlayer were found to be key parameters that determine the structure and performance of the mLbL-assembled membranes. Among the interlayers examined, the PEI/PAA interlayer not only yielded membranes with superior performance but also with a highly smooth surface beneficial for antifouling.