Abstract:chitooligosaccharide (COS) are degradation products of chitin or chitosan, demonstrating good biocompatibility, degradability, non-toxicity, and multiple bioactivities. COS have been widely used in food, cosmetics, composite materials, wastewater treatment, and biomedical industries. Currently, researchers mainly use physical, chemical, and biological enzyme methods to prepare COS. Physical and chemical methods have large limitations, and it is difficult to synthesize the target products with specific requirements in an efficient and green way. Bio-enzymatic preparation of COS shows a mild, controllable, and environmental friendly reaction process, overcoming the drawbacks of physical and chemical methods. The purity of COS can be improved by separation and purification techniques such as membrane separation, gel filtration chromatography, CM-SephadexC-25 ion-exchange column chromatography, and immobilized metal affinity chromatography. This review summarized the research progress in COS preparation using bio-enzymatic technology, aiming to lay theoretical foundation for high-quality industrial COS preparation. It also gave an overview of the structure, properties, and application of COS, contributing for the research on COS preparation and isolation.