A new kind of soluble eggshell membrane protein (SEP) was prepared from eggshell membrane (ESM). The extraction rate of SEP could rise to 90% by two times, basically accomplishing the complete utilization of the whole ESM. Five proteases were employed as hydrolytic enzyme for the preparation of antioxidative peptides from SEP, and the antioxidative activities of the hydrolysates were investigated using a chemiluminescence method. Among the hydrolysates, alcalase hydrolysates with the highest free radical scavenging activity were further separated into three fractions using Sephadex G-25 gel filtration chromatography of the 4 h hydrolysate (SP1, SP2, and SP3). Among these three fractions, SP2 with an average molecular weight of 618.86 Da possessed the strongest fraction of scavenging activity. The IC50 values of the superoxide radicals, hydroxyl scavenging activities, and protective effect on DNA damage caused by hydroxyl radicals generated were 0.10, 0.18, and 0.95 mg/mL, respectively. These results demonstrate that inexpensive ESM waste could be a new alternative in the production of antioxidative peptides.