The interaction of horseradish peroxidase (HRP) with sodium n-dodecyl sulphate (SDS) as an anionic detergent and n-dodecyl trimethylammonium bromide (DTAB) as a cationic detergent was studied at pH 6.4 by microcalorimetry, equilibrium dialysis and spectroscopy. The enthalpy of unfolding of the HRP by surfactants was determined from the calorimetric enthalpy and the enthalpy of binding was calculated from the Wyman and van’t Hoff relations. The transition concentration, [S](1/2), for the denaturation of HRP by SDS and DTAB is enlightened from the enthalpies of unfolding and of binding. Here, it has signified that the [S](1/2) point occurred at the end of HRP-surfactant electrostatic and the start of hydrophobic interactions. The unfolding of protein is mostly completed at the end of electrostatic interactions.