Identification of natural substances with antioxidant properties is ongoing research for addressing issues related to oxidative stress especially attributed to environmental effects. Our previous study demonstrated that Lateolabrax japonicus peptides (LPH), rich in Glu, Gly, and hydrophobic amino acids, exhibited remarkable antioxidant activity in vitro, with though its action mechanism yet to be revealed. Therefore, to assess the in vivo antioxidative properties of LPH, we employed H₂O₂ to generate oxidative stress in Drosophila melanogaster model. Results indicated that LPH significantly prolonged the lifespan of Drosophila subjected to oxidative stress mostly mediated via LPH's enhancement of the antioxidant defense system and intestinal functions. Antioxidant effects were manifested by a decrease in malondialdehyde (MDA) levels, elevated superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, decreased levels of reactive oxygen species (ROS) in intestinal epithelial cells, and the preservation of intestinal length. LPH effectively controlled the excessive proliferation and differentiation of oxidative stress-induced Drosophila intestinal stem cells. At the gene level, LPH upregulated the expression of antioxidant-related Nrf2 genes while concurrently downregulated mTOR expression level. Furthermore, high-throughput 16S rDNA sequencing revealed that the addition of LPH significantly influenced the diversity and abundance of the intestinal microbiota in H₂O₂-induced Drosophila. These findings provide a deeper understanding of the antioxidative mechanism of LPH, suggesting its potential applications in food industry and to be assessed using other in vivo oxidative stress models.