Chito-oligosaccharides (COS) are being used as important functional materials for many applications due to their bioactivities. The aim of this research has been to assess the relationship between the physico-chemical characteristics, average molecular weight (Mw), acetylation degree (DA), polymerization degree (DP) and specially sequence composition determined by MALDI-TOF MS and the antioxidant properties of COS. These oligosaccharides were obtained by enzymatic depolymerization with chitosanase and lysozyme using a specific chitosan and its reacetylated product. The COS fraction below 5 kDa obtained from chitosanase depolymerization showed the highest capacity to scavenge DPPH radicals and to reduce Fe(3+). A correlation was found between the relative amount of molecules with a given A/D (acetylated vs deacetylated units) ratio within the COS and their antioxidant activity, which could be used to predict the antioxidant behavior of a fraction of chito-oligosaccharides.