Quantum dots have many excellent optical properties such as high quantum yield,long fluorescence lifetime,wide excitation spectrum and narrow emission spectrum,tunable emission wavelength and so on,thus have become a newpopular type of fluorescence probes in these years. Quantum-dot-based DNA nanosensor comprising streptavidin-conjugated quantum dots,capture probes with biotin and reporter probes with Cy5 was designed to detect DNA or RNA segments. Capture probes and reporter probes were connected by the target DNA or RNA segments so that quantum dots and Cy5s could be together and FRET (fluorescence resonance energy transfer) could be detected. In the present work,quantum-dot-based DNA nanosensor was combined with ICCD fluorescence microscopy imaging system through the authors’ experiments. Using the total internal reflection fluorescence (TIRF),FRET between quantum dots and Cy5s was recorded by ICCD showing that segments of single-stranded target DNA with 30-base length were detected in solution using DNA nanosensor. When Cy5-ssDNA-Biotins were added into streptavidin-conjugated quantum dots in solution,by real time recording,the FRET efficiency was found to increase with time,which indicated the process of streptavidin-conjugated quantum dots capturing Cy5-ssDNA-Biotins. It was also observed that streptavidin-conjugated quantum dots and Cy5-ssDNA-Biotins could both enter living Chinese hamster ovary cells and have FRET. The process of streptavidin-conjugated quantum dots capturing Cy5-ssDNA-Biotins was detected in the cells as well and Cy5s were photobleached after a long time of irradiation. It has been proved that detecting DNA or RNA segments in living cells with DNA nanosensor is possible.