Chitooligosaccharides (COS) are -1,4-linked homo-oligosaccharides of N-acetylglucosamine (GlcNAc) or glucosamine (GlcN), and also include hetero-oligosaccharides composed of GlcNAc and GlcN. These sugars are of practical importance because of their various biological activities, such as anti-microbial, anti-inflammatory, anti-oxidant and anti-tumor activities, as well as triggering the innate immunity in plants. The reported data on bioactivities of COS used to contain some uncertainties or contradictions, because the experiments were conducted with poorly characterized COS mixtures. Recently, COS have been satisfactorily characterized with respect to their structures, especially the degree of polymerization (DP) and degree of N-acetylation (DA); thus, the structure-bioactivity relationship of COS has become more unambiguous. To date, various green-chemical strategies involving enzymatic synthesis of COS with designed sequences and desired biological activities have been developed. The enzymatic strategies could involve transglycosylation or glycosynthase reactions using reducing end-activated sugars as the donor substrates and chitinase/chitosanase and their mutants as the biocatalysts. Site-specific chitin deacetylases were also proposed to be applicable for this purpose. Furthermore, to improve the yields of the COS products, metabolic engineering techniques could be applied. The above-mentioned approaches will provide the opportunity to produce tailor-made COS, leading to the enhanced utilization of chitin biomass.