In this research, de novo sequencing was innovatively combined with parallel SPOT synthesis for the efficient screening of biological peptides from TCM or seafood: seahorse with synergistic antioxidant and α-glucosidase inhibitory activities, which is promising for postprandial hyperglycemia management. Gastrointestinal digestion mimic and de novo sequencing were sequentially carried out to predict new peptides from seahorse. After bioinformatic analysis using Peptide Ranker, 82 peptides were eventually synthesized by efficient parallel SPOT technique, and Ser-Val-Try-Leu-Gly-Gly-Ser-Leu-Leu (SVWLGGSLL) was screened out as the most efficient peptide with synergistic antioxidant (DPPH radical scavenging activity of 77%) and α-glucosidase inhibitory activity (IC50 = 0.36 mM). Molecular docking was further carried out to illustrate the favorable ligand-receptor interactions formed such as hydrogen bonding and van der Waals force with low binding free energy of -7.8 kcal/mol. Moreover, pharmacokinetic analysis indicated that SVWLGGSLL was unrelated to toxicity with the advantage of gastrointestinal stability.© 2025 European Peptide Society and John Wiley & Sons Ltd.