In this study, a bioactive derivative of chitooligomers (1.0–3.0 kDa), 4-hydroxybenzyl-COS (HB-COS), was synthesized to enhance antioxidant activity. Hence, HB-COS was evaluated for its capabilities against H2O2-induced oxidative stress in human Chang liver cells. It was found that HB-COS possessed the free radical scavenging activity via decreasing the intracellular reactive oxygen species production. Furthermore, HB-COS significantly reduced the oxidation of DNA in a dose-dependent manner. Notably, HB-COS treatment upregulated the gene and protein expressions of antioxidative enzymes and thereby enhancing the intracellular antioxidant mechanisms. In addition, HB-COS treatment caused a remarkable blockade on degradation of inhibitory kappa B alpha (IκB-α) protein and translocation of nuclear factor kappa B (NF-κB). The current study demonstrated that HB-COS effectively attenuated hydrogen peroxide-induced oxidative stress in Chang liver cells by increasing levels of antioxidant enzymes and inhibiting reactive oxygen species generation, DNA oxidation and the NF-κB signaling pathway.