This study investigated the binding mechanism between proteins and hydrophobic adsorbents from a thermodynamics aspect. The proteins investigated here included lysozyme, myoglobin, and alpha-amylase. The binding isotherms were obtained for the proteins with butyl-sepharose and octyl-sepharose resins at various temperatures and salt concentrations. The binding isotherms were then analyzed using the van't Hoff equation, and the binding enthalpy and entropy of each step of the binding process are discussed. Moreover, the effects of ligand chain length and protein characteristics were also investigated. Notably, the preferential interaction model has been adopted in the discussion of the dehydration step in the binding mechanism. The analytical results show that the binding process is entropy-dominated at higher temperature and the amount of released water molecules increases with temperature. Furthermore, the binding enthalpy (OH H) and heat capacity were calculated by the van't Hoff and Kirchoff equations and were compared with the directly measured enthalpy (DeltaH(ITC)) by isothermal titration calorimetry. The analytical results provide useful insights into hydrophobic interaction in biorecognition systems.