This study aimed at elucidating the interaction mechanism between an imprinted polymer and its template in aqueous environment with thermodynamic aspects. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was chosen as a model template to imprint a co-polymer of 4-vinylpyridine (4-VP) and ethyleneglycol dimethacrylate. Equilibrium binding isotherm analysis and isothermal titration microcalorimetry were used to quantify the contribution of enthalpy and entropy to the binding process, identify the nature of the interactions involved and confirm the existence of binding pockets with shape-complementarity to the template. For the binding process of 2,4-D to the imprinted polymer, we postulate three subprocesses: (1) dehydration of the binding pocket and of the 2,4-D, (2) adsorption of 2,4-D, and (3) rearrangement of the water molecules from the dehydration process. We found that binding in aqueous environment was due to the cumulative effect of π-stacking and electrostatic interactions between the template and the functional monomers. At pH<6, entropy is the dominating driving force, while at pH>6 where the highest difference in binding between the imprinted and a non-imprinted reference polymer was observed, the enthalpy change accounts for most of the binding free energy. The developed microcalorimetric method sheds light on the binding mechanism of analyte molecules with imprinted polymers, in particular if the polymers are used in aqueous solvents.