RNA-fluorescence in situ hybridization (RNA-FISH) is widely utilized to detect the specific RNA sequences and its contribution in cells or tissues through the complementary hybridization with the fluorescence-labeled nucleotide probes. Given the weak signal, RNA-FISH would be combined with the specific signal amplification technique to improve the signal-to-background ratio. However, due to the low resolution, the traditional signal amplification technology is difficult to eliminate the high background and can not quantify RNA accurately. It is an obvious obstacle to the application of RNA-FISH. Based on the third generation hybridization chain reaction (HCR v3.0) technology, the half split probes were designed to eliminate the non-specific hybridization background and trigger fluorescence signal amplification. Here, we established the sensitive and specific RNA-FISH based on HCR v3.0 to detect the viral RNA of enterovirus A71 (EV-A71). Furthermore, combining RNA-FISH with immunofluorescence staining technology (IF) and high-resolution confocal laser imaging, we successfully detected and quantified the viral RNA and 3D polymerase of EV-A71 during the viral infection in a single-cell level. We observed that the viral RNA decreased whereas 3D polymerase increased significantly in the late stage of EV-A71 infection. It was apparently different from the traditional quantification by reverse tran-scriptase quantitative polymerase chain reaction(RT-qPCR) and western blot, which were based on the total cells during viral infection. It demonstrates that the new generation of RNA-FISH technology based on hybridization chain reaction can overcome the shortcoming of masking the change of virus composition by the increase or decrease of population cell number, so as to truly reflect the change of virus in a single cell.