This study was divided into two parts: (i) an optimal hydrolysing procedure of chicken liver hydrolysates (CLHs) and (ii) the in vivo antioxidant properties of CLHs via a D-galactose-induced mouse model. A pepsin-to-raw chicken liver mass ratio (1:400, w:w) and 2-h hydrolysing period were chosen to manufacture CLHs based on yield, peptide level and antioxidant effect. Molecular masses of CLHs were lower than 10 kDa. CLH was rich in aspartic acid and glutamic acid, and also contained both manganese and selenium, which are essential cofactors of superoxide dismutase and glutathione peroxidase, respectively. The contents of cadmium, mercury, tin, and arsenic in CLHs were very low and even no detectible. Regarding the in vivo antioxidant activity of CLHs, a dosage of 1.2 g D-galactose kg−1 body weight increased (P < 0.05) 2-thiobarbituric acid reactive substances values and decreased (P < 0.05) glutathione and Trolox equivalent antioxidant capacity values, as well as superoxide dismutase, catalase, and glutathione peroxidase activities in serum and organs of mice. However, the in vivo antioxidant capacities were improved (P < 0.05) by supplementing CLHs.