- Abstract This study presents the preparation procedure and the properties of nanofiltration membranes fabricated by step-by-step assembly on ultrafiltration supports. This procedure allows the covalent binding of molecular layers of alternating monomers, trimesoyl chloride and m-phenylenediamine, to control film growth and surface chemical functionality. The molecular layers are attached directly onto aminated alumina supports, obtained by functionalization of the supports using an aminosilane reagent, for the first time without the need of an anchoring polymeric layer. The membranes are fabricated by performing different numbers of reaction cycles between the two monomers and are characterized along the various cycles 2–6.5 reaction cycles). Surface analyses demonstrate the development of the layered assembly and the attained functionalizations as a function of exposed moiety. Electron microscopy proves that low number of cycles allows achievement of a smooth film, while layer thickness and heterogeneity increase above five step reactions. The water permeance and the salt rejection capabilities of the fabricated membranes are in the range of nanofiltration, with observed rejections of 20–80% and 20–95% for NaCl and MgSO 4 , respectively. The transport properties correlate well with the number of reaction cycles, thus allowing tuning of the membrane permeance and selectivity by adjusting the total number of layers in the assembly.