DNA binding with small molecule plays an important role in the designing of various anticancer drugs with greater efficacy. The five 9-O-imidazolyl alkyl berberine derivatives (BI) of different chain length has been synthesized and fully characterized. The binding study of calf thymus DNA with these newly synthesized berberine derivative was performed using various biophysical techniques. The binding affinity of BI to calf thymus DNA increased with increasing the chain length. The binding constant value obtained from UV-Vis spectral analysis was 1.84x105for BI1, 2.01x105for BI2, 1.51 x 106 for BI3, 3.66 x 106 for BI4, 6.68 x 106. Partial intercalative binding with strong stabilization of the DNA helix was revealed from circular dichroism spectral study and viscosity measurement. From the ITC experiment it was revealed that the bindings of BI1, BI2, BI3, BI4 and BI5 to calf thymus DNA were favoured by a large positive favourable entropy and negative enthalpy change and the highest spontaneity found for BI5. With the increase in chain length the binding was driven by a stronger entropy term with a higher binding constant indicates involvement of hydrophobic force for all these interaction. High binding affinities of calf thymus DNA with berberine-imidazole derivatives might be helpful for new drug design.