To effectively separate Mg2+ and Li+ from salt lake brine with high Mg2+/Li+ mass ratio, a positive charged nanofiltration membrane with abundant -NH3+ and -NH2+ was designed and fabricated via interfacial polymerization between polyethyleneimine (PEI) and trimesoyl chloride (TMC) on the polyethersulfone ultrafiltration membrane. Zeta potential test revealed that at pH below 9.3, the resulting membrane exhibited a high positive charge due to a large amount of unreacted amine groups on the PEI branch. In the separation process, Donnan exclusion effect dominated the separation performance of resulting membranes for Mg2+ and Li+. The results showed that the Mg2+/Li+ mass ratio declined from initial 20 to 1.3 and the separation factor S-Mg.Li was as low as 0.05 with a pure water permeation of 5.02 L/(m(2)hbar) after filtration with resulting NF membrane. The difference between the rejection for Mg2+ and Li+ reached up to 76%. Compared with membrane in previous studies, the separation performance for Mg2+ and Li+ of positive charged NF membrane prepared in this work increased more than one time. This research demonstrated the potential of positive charged PEI-TMC composite NF membrane for the separation of Mg2+ and Li+ from salt lake brine with high Mg2+/Li+ mass ratio.