Owing to its inherent ability to overcome the spectral crosstalk, high sensitivity, and non-destructivity characteristic, fluorescence resonance energy transfer (FRET) quantitative measurement (spFRET) method based on spectral unmixing has been generally regarded as the most promising approach of live-cell FRET measurement. This paper first briefly introduced the quantitative FRET measurement method and the related research advances on FRET technology. Second, the principle, development process, and robustness of spFRET based on linear separation of emission spectra (Em-unmixing) and linear separation of excitation emission spectra (ExEm-unmixing), respectively, were introduced. Finally, the potential advantages of the two spFRET technologies in live-cell FRET applications was also provided and discussed.