Antigen-antibody interaction is an attractive therapeutic and diagnostic target. The antigen binding site is formed by respectively three complementarity-determining regions (CDRs) in the variable regions of heavy (VH) and light (VL) chains. The amino acid sequences of the CDRs determine the specificity and affinity of the antibody against the antigen. Antibody is known to exhibit conformational change in the antigen binding site after forming the initial complex. This structural rearrangement, widely expressed as “induced fit”, is believed to be critical for tight (Kdof nM ranges) antigen-antibody interaction. However, elucidation of this process is limited by availability of biophysical and biochemical technique for characterizing this initial complex. Here, we employed Surface Plasmon Resonance (SPR) method, and obtained kinetic and thermodynamic properties of the interaction between single chain variable fragment (scFv) of 64M5 antibody and oligonucleotides containing a (6-4) photoproduct for the initial and structural rearrangement step, separately. These analyses revealed that the 64M5scFv initially associated the (6-4) photoproduct by the entropy-driven hydrophobic interactions, while in the following structural rearrangement step, the enthalpy-driven interactions, presumably by hydrogen bonds and van der Waals interactions, were formed to tightly and specifically bind the antigen. Furthermore, mutational analyses revealed that the flexibility of the antigen binding site was critical for the second step. It could be assumed that optimization of the flexibility of the antigen recognition site is an important clue for rational design of high affinity antibody.