Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing ∼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing. Display omitted •The mGHG motif can dictate the cleavage site with single-nucleotide precision•Processing site of primary microRNA is determined by DROSHA rather than DGCR8•The dsRBD of DROSHA recognizes the mGHG motif Target specificity of microRNA is determined by DROSHA cleavage sites. Kwon et al. show how DROSHA precisely selects the cleavage sites using the interaction between its double-stranded RNA-binding domain (dsRBD) and the mGHG motif of the primary microRNA.