The optimum saponification extracting conditions of sterol of Pleurotus tuber-regium (Fr.) Sing Sclerotium (PTRSS) with ultrasonic were studied by the Response Surface Methodology (RSM). The results of optimum extraction conditions from Box-Behnken model were as follow: ultrasonic time, 48 min; solid-to-liquid ratio, 1:12.5 g/mL; saponification temperature, 81 ℃; saponification concentration, 0.61 mol/L. Under these conditions, the sterol content was 497.89 ± 0.75 µg/g. Fourier transform infrared spectroscopy was used to analyze the structures of the substance and shows that it has the characteristic peak of sterol. In a certain concentration range, the dose-effect relationship between sterol of PTRSS and antioxidant activity. The binding capacity of 4 mg/mL sterol PTRSS to sodium cholate, sodium glycylcholate and sodium taurocholate in vitro was 36.05 ± 0.76%, 29.41 ± 0.64%, 8.90 ± 0.34%, respectively, indicating that it possessed good hypolipodemic effect in vitro. [ABSTRACT FROM AUTHOR]