The design and development of a water-soluble heterocyclic ligand are believed to be an alternative way for improving the separation efficiency of actinides from lanthanides. Herein, we designed and synthesized a novel hydrophilic multidentate ligand: disulfonated N,N'-diphenyl-2,9-diamide-1,10-phenanthroline (DS-Ph-DAPhen) with soft and hard donor atoms, as a masking agent in aqueous solutions for Am(III) separation. The combination of N,N,N',N'-tetraoctyldiglycolamide in kerosene and DS-Ph-DAPhen in aqueous phases could separate Am(III) from Eu(III) across a range of nitric acid concentrations with very high selectivity. The coordination behaviors of Eu(III) with DS-Ph-DAPhen in aqueous solutions were studied by UV-vis titration, electrospray ionization mass spectrometry, and Fourier transform infrared spectra. The results indicated that Eu(III) ions could form both 1:1 and 1:2 complexes with the DS-Ph-DAPhen ligand in aqueous solution. Density functional theory calculation suggests that there are more covalent characters for Am-N bonds than that for Eu-N bonds in the complexes, which supports the better selectivity of the DS-Ph-DAPhen ligand toward Am(IU) over Eu(III). This work demonstrates a feasible alternative approach to separating trivalent actinides from lanthanides with high selectivity.