Daunomycin (DM) is a clinically used antitumor anthracycline antibiotic, which is transported primarily by human serum albumin (HSA) in the blood. Binding characteristics are therefore of interest for both the pharmacokinetics and pharmacodynamics of DM. A new optical biosensor technique based on the resonant mirrorwas used to characterize interaction ofDMwith HSA at different temperatures and the affinity constants were obtained. The HSA-DM interaction is exothermic with having favorable enthalpy and entropy followed by the integrated van't Hoff equation analysis. Fluorescence studies showed that DM has an ability to quench the intrinsic fluorescence of HSA through a static quenching procedure according to the Stern–Volmer equation and DM displays a pH-dependent binding affinity to HSA. Molecular modeling calculations showed that the DM binds HSA to a non-classical drug binding site and further analysis of the binding site of DM within the HSA molecule suggested that hydrophobic contacts, hydrogen bond formation and electrostatic interactions account for the binding of DM.