In recent years, several high-resolution structures of aptamer complexes have shed light on the binding mode and recognition principles of aptamer complexe interactions. In some cases, however, the aptamer complex binding behavior and mechanism are not clearly understood, especially with the absence of structural information. In this study, it was demonstrated that isothermal titration calorimetry (ITC) and circular dichroism (CD) were useful tools for studying the fundamental binding mechanism between a DNA aptamer and L-tyrosinamide (L-TyrNH(2)). To gain further insight into this behavior, thermodynamic and conformational measurements under different parameters such as salt concentration, temperature, pH value, analogue of L-TyrNH(2), and metal ion were carried out. The thermodynamic signature along with the coupled CD spectral change suggest that this binding behavior is an enthalpy-driven process, and the aptamer has a conformational change from B-form to A-form. The results showed that the interaction is an induced fit binding, and the driving forces in this binding behavior may include electrostatic interactions, hydrophobic effects, hydrogen bonding, and the binding-linked protonation process. The amide group and phenolic hydroxyl group of the L-TyrNH(2) play a vital role in this binding mechanism. In addition, it should be noted that Mg2+ not only improves binding affinity but also helps change the structure of the DNA aptamer.