The authors report a new graphene oxide nanosheet (GONS) based fluorescence method for mRNA assay with duplex-specific nuclease (DSN)-assisted signal amplification. Following hybridization between the target mRNA and its complementary DNA probe, the ssDNA in the DNA/mRNA hybrid is selectively cleaved by DSN to produce small fragments. The released mRNA molecule then initiates another cycle of hybridization and DSN digestion. In this manner, each mRNA molecule can specifically trigger many cycles of hybridization and DNA cleavages to produce numerous small DNA fragments. The short DNA fragments can exhibit strong fluorescence signals due to the weak adsorption of GONS to them. This fluorescence assay for mRNA with increased selectivity has a 1 fM detection limit under optimal conditions. Furthermore, this assay was successfully used for mRNA imaging in situ.