Seed longevity is a vital determinant of plant survival, agricultural productivity, and genetic resource conservation. However, seed viability progressively declines during storage, largely due to oxidative stress. In this study, we investigated the relationships between hydrogen peroxide (H₂O₂) accumulation, peroxidase activity, and germination performance in Arabidopsis thaliana seeds subjected to long-term storage. Seeds were stored at 4 °C in darkness for durations ranging from 23 to 536 days. Biochemical assays were performed to quantify H₂O₂ concentration using the Amplex Red Hydrogen Peroxide/Peroxidase Assay, while peroxidase activity was assessed in the same extracts. Germination frequency was determined on Murashige and Skoog (MS) medium under controlled conditions. Results revealed a pronounced increase in H₂O₂ levels with extended storage, peaking at 354 days, followed by a decline at later stages. This pattern suggests that ROS accumulation initially exceeds antioxidant buffering capacity, triggering oxidative stress and cellular damage, while the subsequent decline reflects metabolic inactivity and extensive viability loss. Peroxidase activity displayed a distinct temporal pattern, with maximum activity at 256 days, preceding the H₂O₂ peak. This indicates an induced but transient antioxidant defense that was not sustained during prolonged storage. Germination rates correlated with these biochemical changes: seeds maintained >90% germination up to 54 days, but declined to ~40–50% at 354 days, and <30% by 536 days. Collectively, our findings highlight the central role of oxidative stress in seed deterioration. The temporal misalignment between ROS accumulation and peroxidase activity underscores the imbalance between oxidative challenge and antioxidant response as a key driver of seed aging and loss of viability.