Benzotriazole ultraviolet stabilizers are widely used as photostabilizers in plastic films, rubber, coatings, synthetic fibers, and electronic products. Their tendency to leach into soil, combined with inherent persistence, bioaccumulation potential, and biotoxicity, has led to their classification as emerging persistent organic pollutants threatening ecosystem integrity. Despite these concerns, knowledge gaps persist regarding their environmental behavior at the soil–biota interface and associated ecological risks. This study examined the bioaccumulation kinetics and toxicological effects of 2-(2H-benzotriazol-2-yl)-4,6-di-tert-pentylphenol (UV-328) in the earthworm E. fetida through controlled soil exposure experiments and high-resolution mass spectrometry analysis. UV-328 exhibited significant bioaccumulation with distinct temporal dynamics, reaching an equilibrium tissue concentration of 9.45 mg/kg and a bioaccumulation factor of 2.35, consistent with its high hydrophobicity (log Kow = 7.3). High-concentration exposure (50 mg/kg) significantly reduced cocoon production and body weight by 38.3% and 30.0%, respectively, indicating dose-dependent reproductive toxicity. Biochemical analysis revealed oxidative stress induction with significantly elevated superoxide dismutase (SOD) activity, while glutathione S-transferase (GST) activity showed a significant early response within 7 days of exposure at all tested concentrations. Integrated Biomarker Response (IBR) analysis indicated preferential activation of detoxification pathways relative to oxidative damage markers. The non-parallel dose–response relationships between oxidative stress biomarkers and reproductive endpoints indicate that the mechanistic link between these responses warrants further investigation. These findings provide baseline ecotoxicological evidence for risk assessment of benzotriazole UV stabilizers in terrestrial ecosystems.