The novel trinuclear Pd(II) complex with an aryl oxime ligand, [Pd₃(C,N-(C₆H₄C(Cl)NO)-4)₆], was synthesized and structurally characterized by elemental analysis (C, H, N), IR, resonance signals in the NMR, and single crystal X-ray crystallography. The interaction ability of the complex with native calf thymus DNA (CT-DNA) was monitored as a function of the metal complex–DNA molar ratio by UV-Vis absorption spectrophotometry, fluorescence spectroscopy, circular dichroism (CD) and thermal denaturation methods. All the experimental evidence indicated that this complex could strongly bind to CT-DNA via a groove mechanism. Further, the albumin interactions of the complex were investigated using fluorescence quenching and synchronous fluorescence spectra. The results of fluorescence titration suggested that the fluorescence quenching of BSA by the complex was a static quenching procedure. The site marker displacement experiment has suggested the location of the complex binding to BSA was Sudlow's site I in subdomain IIA. Finally, the molecular docking experiment confirmed the above results and effectively proved the binding of the Pd(II) complex to BSA and DNA.