Abstract: Cyclic oligonucleotides have important biological functions and application value due to their unique topological structure and enzymatic stability. This review provides a systematic exposition of various synthesis techniques employed for the production of cyclic oligonucleotides, encompassing both enzymatic and chemical synthesis. Due to its gentle reaction conditions, favorable biocompatibility, and specificity, enzymatic synthesis has gained considerable interest. Meanwhile, chemical synthesis enables the cyclization of modified nucleic acid sequences or nucleic acid analogues, and effectively compensates for the shortcomings of enzymatic cyclization. This review also explores recent advancements in the application of cyclic oligonucleotides, such as cyclic aptamers, circular antisense oligonucleotides, cyclic small interfering RNA, and other circular RNAs. These studies underscore the significant potential of cyclic oligonucleotides in the realms of disease treatment and life sciences, offering valuable insights for future investigations.