The temperature dependence of the interactions between some steroids (fusidic acid, ouabain and ethynodioldiacetate) and human serum albumin (HSA) at physiological pH (7.4) were studied systematically by square-wave voltammetry. Also, these interactions were proven by means of UV–Vis. absorption spectroscopy and FT-IR spectroscopy techniques. The interaction parameters (binding constants and stoichiometries) at temperature range of 297.5-312.5 K were determined from electrochemical data. It is worthy that the temperature has played a positive role on the interactions of fusidic acid with HSA; however it has negative effective on the interaction parameters of HSA with ouabain or ethynodioldiacetate.