AbstractIn this paper, we propose a novel and sensitive ratiometric analysis method that uses the fractional intensities of time-resolved fluorescence of genetically encoded fluorescent NADH/NAD⁺ biosensors, Peredox, SoNar, and Frex. When the conformations of the biosensors change upon NADH/NAD⁺ binding, the fractional intensities (α _i τ _i ) have opposite changing trends. Their ratios could be exploited to quantify NADH/NAD⁺ levels with a larger dynamic range and higher resolution versus commonly used fluorescence intensity and lifetime methods. Moreover, only one excitation and one emission wavelength are required for this ratiometric measurement. This eliminates problems of traditional excitation-ratiometric and emission-ratiometric methods. This method could be used to simplify the design and achieve highly sensitive analyte quantification of genetically encoded fluorescent biosensors. Wide potential applications could be developed for imaging live cell metabolism based on this new method.