Phycocyanin (PC) is a protein that possesses a multitude of bioactive components with nutritional and pharmaceutical properties. This study used continuous chromatographic techniques such as ultrafiltration centrifugation, Superdex™ increase 10/300 GL gel filtration chromatography, liquid chromatography-tandem mass spectrometry (LC/MS/MS), and reverse-phase high-performance liquid chromatography (RP-HPLC). The antiallergic peptides from PC were obtained by alcalase hydrolysis and were then screened and identified. F3-III showed strong anti-allergic activity (77.25%) among the components (F3-I-F3-V) obtained by RP-HPLC. Subsequently, a novel peptide comprising six amino acids, designated FPYTTQ, was identified and demonstrated potent anti-allergic properties by inhibiting the release of β-hexosaminic (β-HEX) acid and histamine during IgE-mediated RBL-2H3 cell threshing. Therefore, it can be concluded that the polypeptide FPYTTQ, derived from PC, can be employed as an effective anti-allergic agent and that PC represents a promising source of novel ingredients for the development of anti-allergic foods and pharmaceuticals.