We recently developed a quantitative Förster resonance energy transfer (FRET) measurement method based on emission-spectral unmixing (Iem-spFRET). We here developed an improved Iem-spFRET method (termed as IIem-spFRET) for more robust FRET measurement in living cells. First, two background (BG) spectral fingerprints measured from blank living cells are introduced to remove BG and autofluorescence. Second, we introduce a factor denoting the ratio of two molar extinction coefficient ratios () of acceptor to donor at two excitations into IIem-spFRET for direct measurement of the values using a tandem construct with unknown FRET efficiency (). We performed IIem-spFRET on our microscope–spectrometer platform to measure the values of Venus (V) to Cerulean (C) and the values of C32V, CVC, VCV, and VCVV constructs, respectively, in living Huh7 cells. For the C32V or CVC cells, the Iem-spFRET and IIem-spFRET methods measured consistent values. However, for the cells especially with low expressing levels of VCV or VCVV, the values measured by Iem-spFRET showed large deviations and fluctuations, whereas the IIem-spFRET method greatly improved the measured values. Collectively, IIem-spFRET is a powerful and robust tool for quantitatively measuring FRET signal in living cells.