In this contribution, the interaction between the [(η⁶-p-cymene)Ru(benzaldehyde-N⁴-phenylthiosemicarbazone)Cl]Cl anticancer drug and calf thymus DNA (ctDNA) was investigated systematically by using multispectroscopic and electrochemical methods along with viscosity measurements and a DNA melting technique. The absorption spectra of the drug with ctDNA showed a slight blue shift and weak hypochromic effect. The relative viscosity and the melting temperature of ctDNA all increased after the addition of the drug. Using berberine (BH) as a fluorescence probe, this drug statically quenched the fluorescence of the ctDNA–BH complex, and hydrogen bonding and van der Waals interactions played important roles in such an interaction. All the competitive experimental evidence indicated that the binding mode of the drug with ctDNA was intercalative binding. The influences of ionic strength, chemical denaturants, hyperthermia and pH on the binding of the drug with ctDNA were also investigated. The results of FT-IR and CD spectra indicated that the B-form conformation of ctDNA was constant even after the binding interaction of the drug with G–C base pairs of ctDNA. The results of electrochemical experiments further verified the intercalative interaction between ctDNA and the drug. All these results indicated that the biological activity of ctDNA was affected by the [(η⁶-p-cymene)Ru(benzaldehyde-N⁴-phenylthiosemicarbazone)Cl]Cl anticancer drug dramatically.