To clarify the interaction of phosphine copper(I) complex with DNA, our study reports the synthesis of a new phosphine copper(I) complex, along with a detailed analysis of the geometry characterization and its interaction with double-stranded DNA. The triclinic phase Cu(PPh)(L)(I) with a tetrahedral geometry was identified as the product of the reaction of copper(I) iodide with (E,E)-N,N'-1,2-Ethanediylbis[1-(3-pyridinyl)methanimine] ligand and triphenylphosphine by single-crystal X-ray analysis. Molecular interaction of the synthesized complex with the calf thymus deoxyribonucleic acid (ct-DNA) was investigated in the physiological buffer (pH 7.4) by multi-spectroscopic approaches associated with a competitive displacement towards Hoechst 33258 and methylene blue (MB) as groove and intercalator probes. The fluorescence and UV/Vis results detected the formation of a complex-DNA adduct in the ground-state with a binding affinity in order of 10 M, which is in keeping with both groove binders and intercalators. The thermodynamic parameters, ΔS = -200.31 ± 0.08 cal/mol·K and ΔH = -63.11 ± 0.24 kcal/mol, confirmed that the van der Waals interaction is the main driving force for the binding process. Moreover, the ionic strength and pH effect experiments demonstrated the electrostatic interactions between the complex and DNA is negligible. Analysis of the molecular docking simulation declared the flat (E,E)-N,N'-1,2-Ethanediylbis[1-(3-pyridinyl)methanimine] part of the complex was inserted between the sequential A…T/A…T base pairs, while the phosphine substituents were located in the groove, i.e. threading intercalation. Besides, the cytotoxicity of the complex against the MCF-7 human breast cancer cells was detected at IC50 = 10 μg/mL.