Isothermal titration calorimetry (ITC) was carried out to explore the condensation process of plasmid DNA (pDNA) molecules induced by poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) as a condensing agent. The ITC curves measured can be divided into two distinctive endothermic binding processes: the first was the binding of PEG-PLL to the elongated pDNA, and the second was the binding that accompanied the pDNA conformational transition. The thermodynamic parameters were obtained by fitting each ITC curve using our recently developed fitting method. The binding of PEG-PLL to the pDNA was accompanied by a small increase in enthalpy, a large increase in entropy, and a large decrease in free energy. The binding stabilized as the polymerization degree of PLL on PEG-PLL increased and the salt concentration decreased. Changes in the thermodynamic parameters are discussed in relation to both the polymerization degree of PLL on PEG-PLL and the salt concentration.