Enriched in proteins and with similar amino acid compositions to the terrestrial vegetables, Porphyra has become an important economic marine algae with high nutritional and medical values in China. In order to obtain peptides holding antibacterial activity, Porphyra yezoensis was utilized as raw material and water, NaCl, HCl and NaOH were used as the solvents. Water-dissolved, salt-dissolved, acid-dissolved and alkaline-dissolved proteins were prepared by the extraction, ammonium sulfate salting-out, dialysis and freeze-drying, respectively. The four types of proteins were hydrolyzed respectively by pepsin, papain, neutral protease, trypsin, acid protease and compound protease under the optimum conditions for each enzyme. Twenty-four hydrolysates were selected to evaluate the antibacterial activity to Staphylococcus aureus, Micrococcus tetragenus, Bacillus subtilis and Escherichia coli that were used as the indicator bacteria. Moreover, the proteins were divided into 7 portions by fractional precipitation with different saturation degree of ammonium sulfate; and their hydrolysates were fractioned to peptide fragments with different molecular weights by ultrafiltration. The best antibacterial peptides were evaluated by comparing the inhibitory effects of hydrolysates to the indicator bacteria. The result showed that the zymolyte of water-soluble proteins hydrolyzed by pepsin presented the strongest antibacterial activity. Thus, antimicrobial peptides were achieved via fraction precipitation by ammonium sulfate for the proteins, ultrafiltration for their zymolyte, and the ammonium sulfate saturation at 40%−50%, and the range of molecular-weight was less than 5 kDa. In addition, factors (such as temperature, medium pH and antimicrobial peptides concentration) that affect the antibacterial activities were also studied. The results indicated that the antimicrobial peptides from Porphyra yezoensis possessed good thermal and acid-base stability, which may be candidate for a safe food preservative.