Abstract: Abstract: The purpose of this paper was to investigate the efficient preparation technology of antifreeze protein (AFP) of fish skin from Nemipterus virgatus by protease. At first, the effects of the protein types and the molecular weight of the hydrolysate on the thermal hysteresis activity of the AFP were studied. The yield of the AFP was selected as the evaluation index for the further study. Moreover, the ratio of substrate to enzyme, enzymolysis time, enzymolysis temperature and pH value were evaluated according to the yield of AFP. Then the optimum preparation technology of AFP of fish skin from Nemipterus virgatus by protease was investigated by the response surface methodology. Finally, the thermal hysteresis activity of AFP was confirmed and determined by differential scanning calorimeter. The results showed that the thermal hysteresis activity of AFP obtained by alkaline protease was the highest among alcalase protease, neutrase protease, papain protease and compound protease under the optimal proteolysis conditions. Therefore, the alkaline protease was selected as the best protease for AFP preparation. The proteins in the molecular weight range of ≥5-10 kDa displayed the highest thermal hysteresis activity up to (1.20?0.07) ℃, and the yield of the proteins was selected as the evaluation index. The optimum preparation technology of AFP of fish skin from Nemipterus virgatus by protease was as follows: The ratio of substrate to enzyme was 32:1, enzymolysis time was 147 min, enzymolysis temperature was 50 ℃ and pH value was 9. The yield of AFP was up to 49.25%±1.34% under the optimum conditions. The great linear relationship between the predicted value and actual value indicated that the predicted value of the model could reflect the actual yield of AFP accurately. The result by differential scanning calorimeter indicated the AFP of fish skin from Nemipterus virgatus reduced the temperature of the initial crystallization of the solution compared to bovine serum albumin (BSA). Under the optimal retention temperature condition, the thermal hysteresis activity of AFP of fish skin from Nemipterus virgatus was 1.2 ℃. The enzymolysis method by alkaline protease was a rapid and effective method to prepare AFP of fish skin and the AFP exhibited the high thermal hysteresis activity. These results will provide a certain theoretical basis for the comprehensive development and utilization of fish skin from Nemipterus virgatus.