Efficient Li+ and Mg2+ from natural seawater and salt lakes using nanofiltration is essential for efficient extraction of lithium resources and to address the global energy shortage. The combination of size sieving and the Donnan effect is crucial for optimal selectivity. We proposed a positively charged reactive support layer to design dual positively charged layer to enhance the magnesium-lithium separation. The positively charged support layer was prepared by ammoniating the PVC membrane with PEI, and then reacted with TMC to successfully prepare a dual positively charged layer PA/APVC nanofiltration membrane with high Li+/Mg2+ selectivity. The reactive support layer not only established a covalent bond connection with the PA layer, which increased the stability of the active layer, but also showed a positive charge (23.59 mV at pH=7) after ammonization, which further enhanced the Mg2+ repulsion from 82.57 % to 98.56 %. Furthermore, the highest separation factor achieved was 23.34 when a mixed solution containing Mg2+ and Li+ in a mass ratio of 50:1 was utilized, which was 13.48 times that of commercial nanofiltration membranes. The utilization of a reactive substrate membrane in the fabrication process of the PA/APVC membrane offers innovative prospects for future investigations on magnesium-lithium separation, contributing to the advancement of nanofiltration membranes.