MicroRNAs (miRNAs) are a family of endogenous, small, single-stranded non-coding RNAs. Studies have shown that miRNAs play an important role in many life activities by regulating gene expression. In addition, the expression profile of miRNAs may change when some diseases (such as cancer and autoimmune diseases) occur, miRNAs are expected to become diagnostic or prognostic biomarkers in safety evaluation of drugs. Therefore, accurate detection of miRNA expression is very important for its application. The traditional methods of Northern blotting, microarray, reverse transcription quantitative polymerase chain reaction (qRT-PCR) and some new methods of miRNAs detection, such as nanomaterial based miRNAs detection and nucleic acid amplification, are reviewed, and the advantages and disadvantages of these methods are described.