The authors report on a new gold nanoparticle-based colorimetric assay for microRNA (miRNA). It is based on duplex-specific nuclease (DSN)-assisted signal amplification. Following hybridization between target miRNA and its complementary single-stranded DNA (ssDNA), the ssDNA in the DNA:RNA hybrid is selectively cleaved by DSN to produce small DNA fragments. Target microRNA is thus released and will initiate another round of hybridization and DSN digestion. In this manner, each miRNA target can specifically trigger several cycles of hybridization and DSN cleavages to yield numerous small fragments of DNA oligonucleotides. The short DNA fragments (unlike the uncleaved ssDNA probe) exhibit superior capability for stabilizing unmodified AuNP against salt-induced aggregation to the uncleaved ssDNA probe, with an accompanying color change from blue to red. These findings are exploited in a colorimetric assay for miRNA that has a 10 fmol detection limit.