Abstract The interaction of honokiol with human serum albumin (HSA) was investigated for the first time using target protein as a probe by the methods of fluorescence anisotropy, circular dichroism (CD), Fourier transform infrared (FT-IR) and molecular modeling. Upon binding with HSA, the fluorescence intensity of honokiol decreased regularly with the gradual increasing concentration of HSA. In addition, the value of fluorescence anisotropy suggested that the drug was located in a restricted environment of protein. The FT-IR spectra and CD spectra measurements showed that the secondary structure of the protein was changed by the binding of honokiol to HSA. Furthermore, the study of molecular modeling indicated that honokiol could bind to the site I (subdomain IIA) of HSA and hydrophobic interaction was the major acting force.