This study investigated the antifreeze activities and structures of wheat gluten enzymatic hydrolysates at different degrees of hydrolysis and studied the cryoprotective effects of the hydrolysate with the highest antifreeze activity on fermentation properties of frozen dough. We found that the hydrolysate with an enzymolysis time of 90 min had the highest thermal hysteresis value (0.46 °C) and significantly inhibited growth and recrystallization of ice crystals. This might be due to the differences in molecular weight, amino acid composition, secondary structures, and surface hydrophobicity of the hydrolysates. Peptides with a molecular weight of 500–2000 Da accounted for 41.5% of the hydrolysate at 90 min, and this hydrolysate had greater levels of alanine, leucine, glycine, and glutamic acid residues, β-sheet conformation, and surface hydrophobicity than other hydrolysates. Furthermore, fermentation results indicated that the optimum condition for the highest active hydrolysates to protect frozen dough was 0.5% relative to the flour amount, and 0.5% hydrolysate enhanced the fermentation capacity of the frozen dough. Accordingly, wheat gluten enzymatic hydrolysate hydrolyzed for 90 min can protect dough from damage caused by repeated freezing–thawing. This study offers new insights into the preparation of antifreeze peptides and the use of wheat gluten.