This study aimed to evaluate the effect of immersion freezing (IF) at different temperatures on ice crystal formation and protein properties in fish muscle. Snakehead blocks were frozen by IF at -20, -30, and -40 °C, and conventional air freezing (AF) at -20 °C. The size of ice crystals in the frozen samples was evaluated using Image J software. Changes in protein properties were analyzed by Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). Snakehead blocks frozen using IF contained smaller ice crystals and better microstructures, especially at lower temperatures. The mean cross-sectional areas of ice crystals formed in the frozen samples were 308.8, 142.4, and 86.5 μm for IF treatments at -20, -30, and -40 °C, respectively, and 939.6 μm for the AF treatment. The FT-IR results show that protein aggregation in the frozen fish blocks was manifested by a decrease in α-helices connected to the increased random coil fraction. The DSC results show that samples prepared by IF had a higher denaturation enthalpy () and denaturation maximum temperature () than those prepared by AF. These results confirm that IF generated a larger number of smaller ice crystals, which is conducive to food preservation.