The interactions of lanthanide ions (Ln3+) with bovine serum albumin (BSA) under mimetic physiological conditions (310.15 K, pH 6.7, 0.1MNaCl) were studied by microcalorimetry. For the first time, based on Two Sets of Independent Sites Model, molar enthalpies (ΔrHm1, ΔrHm2) and coordination number (n1, n2) of the two sets of binding sites with different affinity were obtained directly from the microcalorimetric results. It was shown that the interactions are endothermic and entropy-driving processes. By combining with fluorescence spectroscopy, other thermodynamic parameters (ΔrGm1, ΔrSm1) were determined for high-affinity specific sites.