Abstract: To achieve green and efficient extraction of Pleurotus ostreatus protein (PP), the phase diagram of the PEG-(NH₄)₂SO₄ aqueous two-phase system (ATPS) was constructed using the cloud point method. Protein extraction concentration was utilized as evaluation indicator to investigate the effects of ATPS factors such as the molecular weight and mass fraction of PEG, mass fraction of (NH₄)₂SO₄, and proportion of added crude PP extraction. Furthermore, the basic components of PP including emulsification characteristics, foaming properties, and water and oil holding capacities were explored. The optimal process conditions for PP extraction were determined using response surface methodology, with PEG2000 mass fraction set at 19.5%, (NH₄)₂SO₄ mass fraction at 22.6%, and protein crude extract mass fraction at 26%. Under these conditions, the extraction rate of protein reached 93.16%, with a protein content of 1.71 mg/mL. Analysis of the physical and chemical properties revealed uniform protein composition with an average particle size of approximately 220.2 nm. The total amino acid content was measured at 221.46 mg/g, with the highest aspartic acid (Asp) content at 25.06 mg/g. Moreover, PP exhibited exceptional emulsification stability 65.22%, foaming properties 79.87%, and water holding capacity 2.61±0.18 g/g. Compared with similar food proteins, PP protein had higher emulsifying stability, foaming ability, and water holding capacity. This study used a green, environmentally friendly, and low toxicity aqueous two-phase method to extract PP protein, providing a new approach for the green and sustainable development of protein extraction in the food industry, and a research foundation for the development of functional foods based on PP protein.