The binding of doxepin hydrochloride (DH) to human serum albumin (HSA) was investigated by fluorescence, UV–vis absorption and circular dichroism (CD) techniques under simulative physiological conditions. The binding parameters have been evaluated by fluorescence quenching method. Negative enthalpy (ΔH°) and positive entropy (ΔS°) values indicated that both hydrogen bond and hydrophobic forces played a major role in the binding of DH to HSA. The distance r between donor (HSA) and acceptor (DH) was obtained according to the Forster's theory of non-radiation energy transfer. Spectral results revealed that the binding of DH to HSA induced conformational changes in HSA. The effect of common ions on the binding constant of DH–HSA was also examined.