The 2024 Nobel Prize in Physiology or Medicine was awarded to American scientists Victor Ambros and Gary Ruvkun in recognition of their discovery of microRNA (miRNA) and its role in regulating gene expression at the post-transcriptional level. miRNA is a type of small non-coding RNA (ncRNA) that regulates gene expression by binding to messenger RNA (mRNA). It exists not only in model organisms such as Caenorhabditis elegans (C. elegans) but also plays an important role in multicellular organisms, including humans, participating in regulating key life activities such as the cell cycle, cell death, and tissue differentiation. miRNAs have also been found in viruses, where they are involved in the process of viral infection. The discovery of miRNA has not only opened up a new research field in ncRNA but also challenged the classic “central dogma” of molecular biology. This dogma traditionally transcribes the linear transmission of genetic information: from DNA to mRNA, then translated into proteins, which ultimately carry out biological functions. However, due to the competitive binding of miRNAs with mRNA and other ncRNAs in cells, such as long non-coding RNA (lncRNA) and circular RNA (circRNA), a vast and complex gene expression regulatory network, known as the competing endogenous RNA (ceRNA) network, has emerged. The complexity and sophistication of the ceRNA regulatory network offer new perspectives for transcriptome research, aid in the exploration of gene functions and regulatory mechanisms at a deeper level, and then enable a more comprehensive understanding of various biological phenomena. Moreover, a complex interaction and regulatory network exists between miRNA and other ncRNAs. miRNA and other ncRNAs may also be generated through the splicing of the same genes, which have complex transcripts capable of simultaneously producing multiple types of ncRNAs, including miRNA, lncRNA, circRNA, etc., all of which are involved in a variety of biological processes. Meanwhile, miRNA itself is encoded by genes in the genome, and its expression is also regulated by other coding or ncRNAs. Together with mRNA and other ncRNAs, miRNA finely regulates the life activities of cells and affects the physiological and pathological functions of the body. The dysregulation of miRNA expression is closely linked to the onset and progression of many diseases, particularly cancers, cardiovascular diseases, and neurodegenerative disordors. Furthermore, miRNA provides new molecular markers and targets for the diagnosis and treatment of these diseases. In terms of disease diagnosis, miRNA can stably exist in body fluids and serve as a biomarker for many diseases. The research and development of miRNA drugs is currently advancing rapidly. At present, the research and development of miRNA drugs mainly includes endogenous miRNA analogs and inhibitors targeting endogenous miRNA. Although challenges such as stability, immunogenicity, and permeability remain, advances in chemical modification and delivery technologies are gradually overcoming these obstacles, promoting the clinical translation of miRNA-based drugs. This article summarizes the discovery, mechanism of action, and biological functions of miRNAs, as well as their interaction networks with other ncRNAs, and explores the future prospects of miRNA applications.