Tetradentate 1,10-phenanthroline-2,9-dicarboxamide (PDAM) derivatives have been well documented as effective binding ligands toward MA(III) and Ln(III), while the structural analysis and species determination of the complexes are still limited. Herein, we report a combined study on the coordination of Eu(III) with PDAM derivatives using electrospray ionization mass spectrometry (ESI-MS), time-resolved laser-induced fluorescence (TRLIF), and density functional theory (DFT) calculations. PDAM derivatives here involve N,N'-diethyl-N,N'-diethyl-2,9-diamide-1,10-phenanthroline (Et-Et-DAPhen), N,N'-dibutyl-N,N'-dibutyl-2,9-diamide-1,10-phenanthroline (But-But-DAPhen), and N,N'-dihexyl-N,N'-dihexyl-2,9-diamide-1,10-phenanthroline (Hex-Hex-DAPhen). The collision-induced dissociation (CID) test shows that these alkyl-DAPhen ligands coordinate strongly with Eu(III), given that the coordination moieties remain intact during CID. The 1/2 Eu-III-L species was found to be the dominant component for all three ligands, as evidenced by ESI-MS and fluorescence titration. The fluorescence decay results indicate that the hydration numbers of Eu(III) are reduced from 9 to 1 upon complexation, in agreement with the fact that the 1/2 Eu-III-L species are formed, and eight water molecules are exactly replaced by eight donor atoms of two alkyl-DAPhen ligands. In addition, the DFT calculations suggest that the 1:2 Eu-III-L species is more stable than the 1/1 Eu-III-L species and the Eu-oxygen/nitrogen (Eu-O/N) bonds have a dominant ionic character, with the O atoms having stronger electron-donating abilities toward Eu(III) in comparison to the N atoms.