Ruthenium(II) arene complexes with curcumin analogs possessing different structures have attracted huge attentions in several areas. Herein, in vitro interactions between three ruthenium(II) arene complexes with curcumin analogs and calf thymus DNA (ctDNA) were comparably investigated by viscosity measurement, multi-spectroscopic techniques, and electrochemical methods. Three complexes caused the red shift of absorption peak and weak hypochromic effect of ctDNA. These complexes increased the melting temperature but decreased the relative viscosity of ctDNA to different content. Three complexes all intercalated into the G–C base pairs of ctDNA by hydrogen bonding and van der Waals interactions, which did not affect the right-handed B-form helicity of ctDNA significantly. The intercalative binding interactions between three complexes and ctDNA were further proved by electrochemical methods. The polarity and the space steric hindrance effect of these complexes were responsible for their differences in their intercalative binding interactions with ctDNA. Complex 1 with higher polarity and less steric hindrance effect exhibited the strongest intercalative binding ability with ctDNA. These results revealed the molecular mechanisms of the intercalative binding interactions between three ruthenium(II) arene complexes with curcumin analogs and DNA, which provided new insight into the biological applications of these complexes in the future.