Antisense RNA molecule represents a unique type of DNA transcript that comprises 19-23 nucleotides and is complementary to mRNA. Antisense RNAs play the crucial role in regulating gene expression at multiple levels, such as at replication, transcription, and translation. In addition, artificial antisense RNAs can effectively regulate the expression of related genes in host cells. With the development of antisense RNA, investigating the functions of antisense RNAs has emerged as a hot research field. This review summarizes our current understanding of antisense RNAs, particularly of the formation of antisense RNAs and their mechanism of regulating the expression of their target genes. In addition, we detail the effects and applications of antisense RNAs in antivirus and anticancer treatments and in regulating the expression of related genes in plants and microorganisms. This review is intended to highlight the key role of antisense RNA in genetic research and guide new investigators to the study of antisense RNAs.