Abstract: The interaction between cryptotanshinone and human serum albumin (HSA) was investigated under simulated physiological conditions. The analytical results of fluorescence spectra indicated that cryptotanshinone quenched the intrinsic fluorescence of HSA via a static quenching procedure. The values of the binding constant and the numbers of binding sites were calculated at 291, 301, 311 K. According to the thermodynamic parameters, electrostatic force might be main acting force between cryptotanshinone and HSA. The site marker competitive studies indicated that cryptotanshinone was bound at sites Ⅰ on HSA. Circular dichroism spectra showed cryptotanshinone changed the structure of HSA. The binding distance between cryptotanshinone and HSA was determined as 2.86 nm according to the Frster theory, indicating that the static fluorescence quenching of HSA by cryptotanshinone was also a non-radiation energy transfer process.