It is of great significance to improve the quality of frozen dough by selecting yeast antifreeze. The enzymatic hydrolysates from silver carp were prepared by enzymolysis with protamex, and their degree of hydrolysis (%), composition and electrophoresis profiles were analyzed. Moreover, the effects of enzymolysis time and concentration of enzymatic hydrolysates on the survival rate of yeast were also investigated. The results showed that yeast had poor frost resistance, with only 0.69% of survival rate after the first freeze-thaw cycle. Components of hydrolysates prepared by enzymolysis for 15, 30, 60, 120, 240 min were similar, but the products of 15 and 30 min had better cryoprotective effect on yeast. By adding enzymatic hydrolysates of 30 min at the concentration of 4%, the survival rate of yeast was increased to 90.73%, which was 42.88% higher than that of the positive control (glycerol). Tricine-SDS-PAGE analysis indicated protein bands distribution of these hydrolysates were similar, but the band signals were gradually decreases with the prolongation of enzymolysis time, and it was interesting to suggest that the new 13 kDa protein might have important relationship to the cryoprotective activity of enzymatic hydrolysates.