N-Oxide zwitterionic polyethyleneimine (ZPEI), a new kind of aqueous phase monomer synthesized by commercially branched polyethyleneimine (PEI) via oxidation reaction, was prepared for fabrication of thin-film composite (TFC) polyamide membranes via interfacial polymerization. The main factors, including the monomer concentration and immersion time of the aqueous phase and organic phase, were investigated. Compared with PEI-TFC membranes, the obtained optimal defect-free ZPEI-TFC membranes exhibited a lower roughness (3.3 ± 0.3 nm), a better surface hydrophilicity, and a smaller pore size (238 Da of MWCO). The positively charged ZPEI-TFC membranes (isoelectric point at pH 8.05) showed higher rejections toward both divalent cationic (MgCl2, 93.0%) and anionic (Na2SO4, 96.1%) salts with a water permeation flux of up to 81.0 L·m–2·h–1 at 6 bar, which surpassed currently reported membranes. More importantly, mainly owing to N-oxide zwitterion with strong hydration capability, ZPEI-TFC membranes displayed a high flux recovery ratio (97.0%) toward a model protein contaminant (bovine serum albumin), indicating good anti-fouling properties. Therefore, the novel N-oxide zwitterion functionalized positively charged nanofiltration membranes provide an alternative for water desalination and sewage reclamation.