Aptamers are nucleic acid molecules that bind to target molecules with high affinity and specificity, which are obtained using a technique known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Although aptamers are being researched and developed as promising molecular-targeted therapeutic agents, the mechanism of aptamer binding to the target proteins with their high affinity and specificity is not clear. Therefore, structural and biophysical studies are important to know that, and isothermal titration calorimetry (ITC) to study the thermodynamic basis of aptamer-target protein interactions has become very important. Furthermore, physicochemical insights obtained from molecular simulation are also important. Understanding the mechanism of aptamer binding will contribute to the development of the aptamer therapeutic agents.