Nucleic acids are an important target for many therapeutics. Small molecules that bind to nucleic acids are important in many aspects of medicines, particularly in cancer chemotherapy. In recent years, many studies have utilized polynucleic acids with various sequences to demonstrate the binding mechanism of daunomycin, a potent anticancer drug. This study describes that isothermal titration calorimetry is a useful tool for studying the fundamental binding mechanism systemically. The results suggest that the binding free energy is more favorable when the temperature is increased. The binding entropy contributes to this effect. Furthermore, the amine group on daunomycin contributes electrostatic interaction that induces the binding process. In addition, enthalpy-entropy compensation is also exhibited in the daunomycin-DNA binding mechanism. This study used an easy, convenient method of performing a systemic study in a recognition system. The results from this study provide additional information about microscopic mechanisms for molecular design and molecular recognition.