Herein the manuscript, we report the interaction of an important drug, Imiquimod (IMQ), with Calf Thymus Deoxyribonucleic Acid (ct-DNA). Despite of being used IMQ in multiple medicinal applications such as immune response modifier, antitumor and antiviral drug its interaction with DNA is not studied yet. To understand their interaction and characterize the binding mechanism between groove and intercalation modes, we used various spectroscopic tools. Fluorescence quenching, time-resolved emission, steady state anisotropy technique, ethidium bromide (EtBr), and 4′,6-diamidino-2-phenylindole (DAPI) displacement assays provided us biophysical aspects of IMQ-DNA interactions. All the above studies revealed the binding mode as intercalation mechanism. Circular Dichroism (CD) also well corroborated the intercalation of IMQ into DNA. Thermodynamic parameters of these bindinginteractions were interpreted with the help of temperature dependent spectroscopic studies characterizing the interaction as enthalpy driven. Further, the binding interactions is explored with the help of molecular docking studies. Based on experimental and computational studies it was concluded that the drug binds to DNA via intercalation mode in AT-rich DNA sequences. These biophysical investigations not only improve our comprehension of IMQ's therapeutic effectiveness but also clarifies the sequence dependent binding processes underlying comparable drug-DNA interactions.