The physicochemical characteristics and functional properties of the Pine pumila nut proteins prepared by the alkali solution method, ultrasonic-assisted method and ultrasonic-vacuum combined method were compared and analyzed. The analysis results showed that the extraction rate of Pine pumila nut protein prepared by ultrasonic-vacuum combined technology was 81.33%, which was higher than those of the alkaline solution method and ultrasonic-assisted method. The main bands of the protein molecules prepared by the three methods were 10.26, 14.35, 23.42, 23.85, and 38.17 ku; The amino acid content was higher than the value recommended by FAO/WHO. The total amino acid contents of the Pinus pumila nut proteins prepared by the alkaline solution method, ultrasonic-assisted method and ultrasonic-vacuum combined method were 74.60, 63.80 and 64.92 g/100 g, respectively, with abundant glutamic acid and arginine. The Pinus pumila nut protein prepared by the ultrasonic-vacuum combined technology was superior to those prepared by the alkali dissolution method and ultrasonic assisted method in terms of water absorption, solubility, emulsifying property, emulsion stability, foamability and foam stability, with water absorption particularly prominent. When the pH was at 10, the maximum water absorption of pine nut protein prepared by the ultrasonic-vacuum combined technology was 4.10 g/g. This study has developed an extraction method with a high extraction rate and a capacity to produce Pinus pumila nut protein with desirable functional properties, which provides a reference for the development and utilization of Pinus pumila nut protein.