We developed a singlewavelength excitationbased quantitative EFRET measurement method (SDWEFRET). This method only needs to collect fluorescence intensities of donor channel (IDD) and FRET channel (IDA) under donor excitation, and can realize rapid FRET imaging in living cells. Combining the dualchannel fluorescence microscopic imaging system, SDWEFRET method does not need any mechanical switch, and the speed of quantitative SDWEFRET imaging only depends on the speed of CCD camera. Therefore, SDWEFRET is applicable to mapping the dynamics of intracellular process in live cells in real time. We performed quantitative SDWEFRET measurement on our dualchannel fluorescence microscope to measure the FRET efficiency of C5V and C17V in living cells, and obtained consistent results with those measured by other methods.