Abstract: Antifreeze proteins (AFPs) can regulate the growth behavior of ice crystals and significantly improve the quality of frozen foods. However, the low content of natural AFPs in living organisms, the difficulty in extraction and purification, and the high production cost greatly limit the development of antifreeze proteins. Therefore, it is important to explore appropriate preparation methods to increase the yield of AFPs and reduce the production cost. In this study, based on the amino acid sequences of carrot AFP, Epinepheluscoioides AFP, and Tenebriomolitor AFP from the National Center for Biotechnology Information (NCBI) database, three rAFPs gene sequences were synthesized by codon optimization, and yeast expression vectors were constructed and multiple screening was performed by G418 in 24-well plates.Three recombinant Pichia pastoris strains producing rCaAFP, rFiAFP and rTmAFP were obtained. The fermentation process of the three recombinant Pichia pastoris strains to produce rAFPs was preliminarily optimized.The concentrations of rCaAFP, rFiAFP and rTmAFP in the fermentation supernatant of the three recombinant Pichia pastoris strains were 214.29, 418.18, and 57.95 mg/L, respectively, after high density fermentation in a 7L fermenter.The production intensities were 1.91, 3.27 and 0.45 mg/L·h, respectively. Then, rCaAFP, rFiAFP and rTmAFP were purified by ultrafiltration centrifugation, ammonium sulfate precipitation, ice affinity adsorption, and high performance liquid chromatography (HPLC). The thermal hysteresis activities of the three rAFPs were 1.62℃, 4.23℃ and 7.57℃(10 mg/mL), respectively. In summary, rCaAFP, rTmAFP and rFiAFP produced by heterologous expression of Pichia pastoris showed high yield, easy purification and good antifreeze activity, which is of great value for the study and application of antifreeze proteins.