Abstract: Procambarus clarkii is highly susceptible to quality deterioration during frozen storage due to its high moisture content and endogenous enzyme activity, with myofibrillar protein (MP) serving as the key determinant of gelation performance and final product quality. To investigate the effects of different freezing temperatures on the physicochemical properties and structure of crayfish myofibrillar protein (MP), this study used fresh crayfish as raw material. MP was extracted after freezing at -18 °C, -40 °C, and -80 °C for 30 days. Key indicators including turbidity, solubility, total sulfhydryl content, surface hydrophobicity, particle size, zeta potential, and Ca²⁺-ATPase activity were systematically measured. Molecular structural characteristics were analyzed via SDS-PAGE electrophoresis, circular dichroism spectroscopy, and endogenous fluorescence spectroscopy to comprehensively evaluate the effects of different freezing temperatures on myofibrillar protein physicochemical properties and structure. Results indicated that compared with fresh samples, MP from the -18 °C frozen group exhibited a 20.91 FTU increase in turbidity, a significant 119.48% rise in surface hydrophobicity, and a 2.78% decrease in total sulfhydryl content. The -40 °C frozen group exhibited the smallest changes in all indicators, with Ca²⁺-ATPase activity decreasing by only 12.5% and α-helix content decreasing by 15.3%, significantly outperforming other frozen groups (P<0.05). Structural analysis revealed that primary structure of MP remained intact in all frozen groups. However, secondary structure showed reduced α-helix content by 15.1%-27.5% and increased β-sheet content by 8.2%-15.6%. This study confirms -40°C as the optimal freezing temperature, providing theoretical support for crayfish product processing.