Nanofiltration membranes (NFMs) have been widely used in desalination processes. However, low permeance often exists because of the compact separation layers. Herein, we demonstrate a novel and versatile approach of interfacial polymerization (IP) to fabricate polyamide (PA) NFMs for fast desalination. Copper hydroxide nanostrands (CuNS) are synthesized and assembled with sodium polystyrene sulfonate (PSS) to form CuNS/PSS as sacrificial interlayers before fabricating PA layers on polyvinylidene fluoride (PVDF) substrates. The CuNS/PSS interlayers can assist to store abundant piperazine (PIP) monomers and regulate the distribution by the charge-driven assembly, which slows the IP reaction by impeding diffusion of PIP monomers into oil-water interfaces. Consequently, wrinkled defect-free PA layers can be formed directly on large-porous substrates after dissolving the sacrificial interlayers. The obtained PA NFMs exhibit excellent separation performances, e.g., competitive water permeance of 20.3 L m−2 h−1 bar−1 and high Na2SO4 rejection of 98.3%. This negative charge-driven assembly strategy introduced in IP process can have great potential for NFMs fabrication.