In this study, the interaction between the phosphodiesterase-3 enzyme inhibitor drug milrinone and biomolecules was investigated by electrochemical, fluorescence spectroscopy, and molecular docking studies for the first time. The interaction between milrinone and biomolecules was investigated according to the decrease in deoxyguanosine oxidation signals of milrinone and calf thymus double-stranded deoxyribonucleic acid (ct-dsDNA) by cyclic voltammetry and differential pulse voltammetry. In fluorescence spectroscopy studies, a competitive study was conducted on ct-dsDNA by adding a well-known fluorescent methylene blue and ct-dsDNA solution. The fluorescent results showed that milrinone had a higher affinity for ct-dsDNA binding compared to methylene blue. Interaction studies show that milrinone binds to ct-dsDNA via a groove-binding mode, and the binding constant values were calculated as 4.27 × 106 M-1 and 6.03 × 104 M-1 at 25 °C, based on cyclic voltammetry and spectroscopic results, respectively. As a result of the interaction of human serum albumin and milrinone, the binding coefficient was calculated as 4.11 × 106 M-1 by cyclic voltammetry. In addition, experimental results were confirmed by obtaining information about the possible spatial structure of the aggregate formed through theoretical calculations based on energy minimization for milrinone- ct-dsDNA and milrinone-human serum albumin mixtures with molecular insertion.