Thermo-dependent changes in the secondary structure of the epithelium and stroma of benign prostatic hyperplasia (BPH) were investigated by Fourier transform infrared (FT-IR) microspectroscopy combined with differential scanning calorimetry (DSC). This microscopic FT-IR/DSC system was used to simulate transurethral thermotherapy for BPH in vitro. The results indicate that thermal-induced conversion from an α helix to a β structure was found in the epithelium and stroma of BPH. However, this thermal-induced conversion behavior in the stroma was less sensitive than that in the epithelium during thermal treatment. The different thermal response between the epithelium and stroma of BPH might be due to the different constitutions of the epithelium and stroma of BPH.