Abstract: Phlebopus portentosus, as an edible fungus with significant health-care value, contains a variety of bioactive substances. Ergosterol, as an important component of bolete mushrooms, has functions such as anti-tumor, anti-viral, antibacterial, immune regulation, anti-inflammatory, and inhibition of lipid accumulation. This study aims to extract and purify ergosterol from Phlebopus portentosus and characterize its structure. On the basis of single-factor experiments, response surface optimization experiments were carried out to determine the optimal extraction process of ergosterol. Fourier transform infrared spectroscopy（FT-IR）, ultraviolet spectroscopy（UV）, nuclear magnetic resonance（NMR）, and high-resolution liquid chromatography-mass spectrometry（LC-MR） were used to analyze and characterize its structure. The results showed that the optimal extraction process of ergosterol from Phlebopus portentosus was as follows: the extraction solvent was methanol, the particle size was 80 mesh, the solid-to-liquid ratio was 1∶ 30（g/mL）, the ultrasonic temperature was 40 ℃, the ultrasonic time was 30 min, and the centrifugation speed was 6 000 r/min. Under these conditions, the yield of ergosterol was 0.528 3%. The analysis of the separated and purified ergosterol by high-performance liquid chromatography showed that there was only one chromatographic peak, and the purity of ergosterol was ＞95%. Through the analysis of FT-IR, UV, ¹H nuclear magnetic resonance, ¹³C nuclear magnetic resonance spectrum and LC-MR of the product, itsmolecular formula was determined to be C₂₈H₄₄O, which was consistent with the structural formula of ergosterol. Its structural formula was further identified by NMR. This study obtained the optimal process for the ultrasonic-assisted extraction of ergosterol from Phlebopus portentosus and determined its structure, providing a reference for the subsequent utilization of ergosterol from Phlebopus portentosus.