In this study, different ultrasound frequencies, including 20 kHz, 23 kHz, and 20/23 kHz, were applied for the treatment of myofibrillar protein (MP) with different protein concentrations (20, 40, and 60 mg/mL). The structures and emulsifying properties of the treated and untreated MPs were compared. The MP structural changes were confirmed based on the active sulfhydryl content (ASC), surface hydrophobicity (SUH), particle size, and turbidity, and the degree of change decreased in the order of 20/23 kHz > 20 kHz ≈ 23 kHz > control (CON). Moreover, the MP emulsifying properties, determined based on the emulsifying activity index (EAI), emulsion stability index (ESI), and creaming stability, were in accordance with the structural changes. Notably, frequency superposition provided more external force through the resonance of cavitation bubbles to induce stronger stress response of myosin in MP, thereby promoting the change of protein structure and emulsifying properties. Results implied that MPs treated with different ultrasound frequencies, especially the superposition of two frequencies, have the potential for wide applications in emulsified-type meat products. [ABSTRACT FROM AUTHOR]