Abstract: To explore the effects of various pretreatment methods on the extraction, separation, and processing properties of Chlorella protein, freezing, freeze-drying, and spray-drying were applied. Proteins were subsequently extracted by high-pressure homogenization and separated using chitosanase combined with ethanol precipitation, followed by determination of extraction yield, purity, amino acid composition, and molecular weight distribution. The protein isolates underwent different storage conditions-refrigeration, freezing, hot-air drying, and freeze-drying-after which their functional properties were evaluated. Results indicated that spray-drying significantly improved the protein extraction rate to 67.3% and increased purity to 48.8%, outperforming both freezing and freeze-drying while better preserving RubisCo and essential amino acids, with total amino acids reaching 74.8% and essential amino acids accounting for 40.8%. Refrigerated storage resulted in superior functional performance, yielding an emulsifying activity index (101.4 m²/g) three times higher than that of whey protein, along with significantly improved solubility (52.9%) and foaming capacity (137.5%). These findings demonstrate that spray-drying enhances extraction efficiency through effective cell wall disruption, while refrigeration maintains structural stability, reduces hydrophobic group exposure, and optimizes functional properties, providing a theoretical basis for efficient extraction and industrial application of Chlorella protein.