Abstract: Diatoms are regarded as ideal chassis organisms for synthetic biology, and their metabolic pathways involved in the biosynthesis of high-value compounds, such as polyunsaturated fatty acids and pigments, have long been major research focuses in algal biology. However, synthetic modification and metabolic pathway reconstruction often disrupt photosynthetic electron transport and induce excessive accumulation of reactive oxygen species, which severely restrict cell growth and biomass productivity in engineered algal strains. To expand the metabolic engineering potential of diatoms as chassis organisms for synthetic biology by providing novel targets for reactive oxygen species regulation, this study systematically identified nine peroxidases in Phaeodactylum tricornutum and determined their subcellular localization. Transcriptomic analysis revealed that the gene encoding the chloroplast stroma-localized ascorbate peroxidase 1 (APx1) was highly expressed, and APx1 knockout mutants were successfully generated using the CRISPR/Cas9system. Compared with the wild type, the APx1 mutants exhibited no obvious growth defects but showed a significant reduction in non-photochemical quenching (NPQ) capacity. Further analyses demonstrated that disruption of APx1 impaired the ascorbate–glutathione cycle, leading to excessive peroxide accumulation. Elevated peroxide levels further suppressed the expression of NPQ-related photoprotective genes, thereby weakening the overall stress tolerance of diatom cells. Taken together, these results demonstrate that APx1 plays a crucial role in peroxide detoxification and serves as an important regulatory target for stress resistance in P. tricornutum.