Nanofiltration membrane with fine-tuned pore structure and inner charge can achieve the efficient extraction of Li resource from Salt Lake. Herein, we synthesized three water-soluble dendrimers with 128, 256, and 512-aromatic amine groups that enable to be protonated to show positive charge. Protonated dendrimer porous intermediate layer, thus can be designed and fabricated via diazotization-coupling reaction, which imparts polyamide dense layer made by interfacial polymerization with optimized screening pore structure and decreased inner negative charge. Enhanced water/salt permeance was obtained due to the formation of nanostripe structure and thinner thickness of separation layer, as high as 360.87 kg m−2 h−1 MPa−1 for 2000 ppm LiCl. Moreover, the asymmetric polyamide nanofilm with decreased negative charge density provided 11.52 times of Li+/Mg2+ separation selectivity compared to the pristine counterparts. For example, the PSF-160A-PA membrane exhibited a Li+/Mg2+ selectivity of 45.8. Higher cut-off lines between water permeance and Li+/Mg2+ separation selectivity were achieved, while achieving higher Li purity and Li recovery was observed, which was better than some of literature-reported membranes. Such work provides a scalable strategy to fine-tune the structural feature of intermediate layer, then form defect-free polyamide nanofilm for accurate ion sieving.