Horseradish peroxidase (HRP) is a classical heme-containing protein which has been applied in many fields. The prosthetic group heme in HRP, especially in unfolded state, can react with hydrogen peroxide (H2O2) to produce a fluorescent product with the maximum emission wavelength at 450 nm. Utilizing this emission band as a fluorescence probe, the unfolding process of HRP in urea can be assessed quantitatively, and the calculated thermodynamic parameters are consistent with those determined by circular dichroism (CD) at 222 nm and steady-state tryptophan (Trp) fluorescence methods. (C) 2016 Elsevier B.V. All rights reserved.