Abstract: The proteins were extracted from the Flammulina velutipes stembase by using alkali, ultrasonic-assisted alkali, colloid mill-assisted alkali and freeze-thaw-assisted alkali methods to investigate the effects of different extraction methods on the functional (water/oil-holding capacity, foaming and foaming stability, emulsification and emulsification stability, solubility), physicochemical (potential, particle size, surface hydrophobicity, sulfhydryl groups) and structural (infrared spectra, fluorescence spectra, secondary structure) properties of the Flammulina velutipes stembase proteins, in order to provide a theoretical basis for the co-utilisation of edible mushroom by-products. Differences between the properties of the Flammulina velutipes stembase proteins extracted by different physically assisted methods. The results showed that ultrasonic-assisted alkali extraction and colloid mill-assisted alkali extraction could significantly (P<0.05) increase the extraction rate and improve the functional properties of the Flammulina velutipes stembase proteins, and significantly (P<0.05) improved the emulsification stability and foaming stability. The β-fold content in the secondary structure was significantly (P<0.05) reduced and the β-turn content was significantly (P<0.05) increased, and these changes in structural properties were closely related to the improvement of physicochemical properties such as surface hydrophobicity and stability. The combination of these two extractions gave the highest extraction rate, but the excessive shear force resulted in a significant reduction in the functional properties of the protein, such as emulsification and foaming. The freeze-thaw extraction gave a relatively low extraction rate and reduced the surface hydrophobicity and sulfhydryl content of the Flammulina velutipes stembase proteins.