Chiral separation is an important issue in pharmaceutical research and industries, because most organic compounds and biological molecules, including many drugs and food additives, are chiral compounds. DNA aptamers are a new group of chiral selectors; however, there still exists deficiencies in the understanding of the molecular basis of their chiral recognition. Herein, a comparative study of the DNA aptamer binding with L-argininamide (L-Arm) and its enantiomer (D-Arm) is investigated by spectroscopic and calorimetric methods. The effect of various experimental conditions such as temperature, pH and salt concentration on the L-Arm and D-Arm binding properties was studied in order to provide information about the chiral recognition mechanism of the DNA aptamer. An isothermal titration calorimetry study reveals that both L-Arm and D-Arm binding with the aptamer are enthalpy driven and entropy cost processes. The protonated amino group of both L-Arm and D-Arm participates in electrostatic interaction and this interaction is stronger for D-Arm than L-Arm binding with the aptamer. From the opposite behavior of the heat capacity change of the two enantiomers, we could suggest that L-Arm and D-Arm bind at different binding sites of the aptamer, resulting in different conformations of the binding complexes. In the binding mechanism, electrostatic interaction provided by the protonated amino group with the aptamer and the conformational change of the nucleic acid upon binding are major processes involved for chiral recognition in the DNA aptamer. This study provides information on chiral separation of D- and L-argininamide by the aptamer, which can be successfully achieved by varying the operation temperature based on the opposite heat capacity dependence of the enantiomers binding with the DNA.