Protamine is a promising natural preservative protein, and the extraction process can affect the physicochemical properties and function of the protein. In this study, ultrasound-assisted (UA) acid extraction was used to extract Coregonus peled protamine (CPP). Changes in the extraction, physicochemical and functional properties of CPP with ultrasound time (10, 20, 30, 40 and 50 min) were investigated. The Peleg model was used to describe the extraction kinetics. The results showed that 30 min UA treatment resulted in the highest protein yield (6.81%) (P ≤ 0.05). In addition, the secondary and tertiary structures of CPP were modified, the content of α-helix and β-sheet increased, the content of β-turn decreased, and the fluorescence intensity, solubility and surface morphology were altered. In addition, the degree of oxidation, bacteriostatic and biological activities of CPP were also affected. In conclusion, the study suggests that acidic UA extraction for a moderate time could potentially increase the protein yield of CPP and improve its functional properties.