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Effective characterization and imaging of endogenous RNA transcripts have important value in the diagnosis, treatment, and prognosis of diseases. Traditional qRT-PCR as a liquid-based RNA detection method might lead to false-negative results due to the admixture of too many nontarget cells. Also, many in situ RNA imaging methods were hindered by long turnaround time and insufficient signals. Here, we describe and evaluate a CRISPR/dCas9-MS2-based RNA fluorescence in situ hybridization assay (RCasFISH) for in situ amplified imaging and quantification of RNA transcripts in fixed cells as well as formalin-fixed, paraffin-embedded (FFPE) tissue sections at a single-molecular level in individual cells. Compared to single molecular FISH (smFISH), RCasFISH yields brighter dot signals and a better signal-to-noise ratio (SNR) with lower costs and less than 1.5 h of hybridization. In addition, by using human epidermal growth factor receptor 2 (HER2) as a model, we quantified individual HER2 mRNA molecules in clinical breast cancer FFPE tissue sections and demonstrated its potential to resolve FISH-equivocal cases. Therefore, RCasFISH may provide a new approach for gene expression studies in basic research and hold the potential for molecular diagnostic applications.