In this study, an electrochemical DNA biosensor was developed using a straightforward methodology to investigate the interaction of indinavir with calf thymus double-stranded deoxyribonucleic acid (ct-dsDNA) for the first time. The decrease in the oxidation signals of deoxyguanosine (dGuo) and deoxy-adenosine (dAdo), measured by differential pulse voltammetry, upon incubation with different con-centrations of indinavir can be attributed to the binding mode of indinavir to ct-dsDNA. The currents of the dGuo and dAdo peaks decreased linearly with the concentration of indinavir in the range of 1.0-10.0μg/mL. The limit of detection and limit of quantification for indinavir were 0.29 and 0.98μg/mL, respectively, based on the dGuo signal, and 0.23 and 0.78μg/mL, respectively, based on the dAdo signal. To gain further insights into the interaction mechanism between indinavir and ct-dsDNA, spectroscopic measurements and molecular docking simulations were performed. The binding constant (Kb) between indinavir and ct-dsDNA was calculated to be 1.64 × 108 M-1, based on spectrofluorometric measure-ments. The obtained results can offer insights into the inhibitory activity of indinavir, which could help to broaden its applications. That is, indinavir can be used to inhibit other mechanisms and/or hallmarks of viral diseases.