MicroRNAs (miRNAs) are endogenous single-stranded RNAs of 18~25 nt in eukaryotic organisms, which can regulate the complementary mRNAs at the post-transcriptional level through cleavage or translational repression of the mRNA targets. Recent years, several hundred miRNAs from animals and plants have been identified. These small modulatory RNAs are cleavaged from a precursor of 60~200 nt RNA hairpin. In animals, the primary transcripts of miRNA genes (pri-miRNAs) are recognized and cleaved into precursor miRNAs (pre-miRNAs) soon by an RNase Ⅲ family nuclease, Drosha; then, pre-miRNAs are transported from the nucleus to the cytoplasm. Once in the cytoplasm, pre-miRNAs are recognized and processed into their mature form by another RNase Ⅲ , Dicer. The procedure is briefly summarized, and the biogenesis of plant miRNAs is also discussed. Further research on the pathway of miRNA maturation can help us to know the mechanism of these small RNAs acted as important regulators, and can investigate their critical roles during development and disease.