The dominant market for lithium-ion batteries (LIBs) in China has driven lithium chemicals with foreseeable growth in demand. Lithium extraction from salt-lake brine in China with high Mg²⁺/Li⁺ ratio has become one of the key challenges in the separation of Mg²⁺ and Li⁺. In this study, we introduce here an unprecedented, highly permselective nanofiltration hollow fiber membrane prepared by Layer-by-Layer (LBL) deposition as a novel alternative for Mg²⁺/Li⁺ separation. Poly(styrene sulfonate) and poly(allylamine hydrochloride) were deposited alternatively on a tailor-made ultrafiltration hollow fiber support and crosslinking with Glutaric dialdehyde. The pure water permeability of LBL NF membrane was 18.4 L m⁻² h⁻¹ bar⁻¹. Under the synergistic effect of size sieving and Donnan exclusion effect, it also achieved promising results when tested with simulated brine. The rejection rate of Mg²⁺ was as high as 99%, and the rejection rate of Li⁺ was as low as −60%, with a separation factor of 74 was observed, setting a record of permselectivity for Mg²⁺/Li⁺ separation for engineerable membranes. This research has paved a new road for the engineering application of LBL nanofiltration membranes for mono and bivalent ion separation.