A series of amphoteric polyurethane surfactant (APU) with long side chain alkyl and side chain carboxyl, as well as the structure of polyether (EO) embedded in main chain were synthesized, which was mainly based on H-MDI, PAE and DMPA. The properties were measured by infrared spectroscopy (FTIR), surface tension, critical micelle concentration, laser-particle size analyzer and electrical conductivity. The study indicated that when the PAE to DMPA ratio was 1:1, APU-2 with excellent comprehensive performance was obtained. The critical micelle concentration of APU-2 was 32.26 mg/L and the surface tension could be as low as 40.42 mN/m. Additional, the isoelectric zone of amphoteric polyurethane was in the region of pH 3.5～5.5 . With a narrow isoelectric zone distribution, APU-2 can be used in waterborne dispersions of a wide pH range.