Layer-by-layer (LBL) assembly is a versatile technology with the ability to produce charged thin film active layers by absorbing oppositely charged polyelectrolytes or nanomaterials through various interactions, which has been proven to be a promising method for preparing separation membranes with desired properties. Recently, nanocomposite membranes fabricated by incorporating various kinds of nanomaterials through the LBL technique have gained increasing interest due to their excellent membrane performances in terms of improved permeability, selectivity, anti-fouling, chlorine resistance, and long-term stability. This review aims to provide a comprehensive investigation of the state-of-the-art achievements of the nanocomposite membranes prepared by LBL assembly. Different LBL assembly methods such as dip coating, spray coating, spin coating, inkjet printing, electric field, and high gravity technologies are introduced. The detailed membrane fabrication processes and their applications in different separation areas including nanofiltration, reverse osmosis, ultrafiltration, microfiltration, pressure retarded osmosis, forward osmosis, pervaporation and organic solvent nanofiltration are summarised and discussed. The advantages and challenges of the LBL nanocomposite membranes are also addressed. Overall, this review provides some fundamental clues for the exploration of LBL assembly techniques for the preparation of separation membranes with preferable performances and applications.