Oxidative stress is associated with retarded growth and the initiation and progression of diseases in pigs. The carboxymethylation of chito-oligosaccharide (COS) can improve its antioxidant activity for such issues. Herein, an orthogonal experiment L9 (3<sup>4</sup>) test design was used to optimise the preparation conditions of carboxymethyl (CM)-COS. Fourier-transform infrared analysis evidenced the carboxymethylation of COS, and an in vitro study indicated that the hydroxyl radical scavenging ability of CM-COS is superior to that of COS. CM-COS can also better promote the absorption of cysteine and increase the expression of the amino acid transport system b<sup>0,+</sup> in intestinal porcine epithelial cells. The results suggested that CM-COS can effectively resist oxidative damage by promoting cysteine absorption mediated by transport system b<sup>0,+</sup>, which provides important information regarding the antioxidative damage application of COS and CM-COS in the pig farming industry.