Abstract: MicroRNA (miRNA) is a class of non-coding small RNAs with significant regulatory functions, capable of influencing various biological processes by modulating post-transcriptional translation. In recent years, the application of miRNAs as biomarkers in in vitro diagnostics (IVD) has garnered considerable attention. This article first introduces the generation and functional mechanisms of miRNAs, and outlines the roles and potential of miRNAs as biomarkers for various diseases, drawing on recent research findings. Subsequently, five traditional miRNA detection techniques are briefly introduced, summarizing their advantages and limitations in clinical diagnostic applications, and two emerging techniques used in miRNA research are enumerated. Finally, the article analyzes the challenges faced by using miRNAs as biomarkers in current clinical diagnostics, addressing four aspects: intended use, reference intervals, testing procedures, and analytical performance.