Autofluorescence imaging has been widely applied as advanced noninvasive diagnostics for in vivo and ex vivo tissues. The optical redox ratio (ORR), which is defined as the fluorescence intensity ratio between reduced nicotine adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), has been used as a diagnostic parameter strongly, because NADH and FAD play an important role in energetic and respiratory metabolism as coenzymes. The ORR method has provided successful assessment in cancer diagnosis including breast, cervical, and oral cancer; few studies have been reported about optical and chemical interference between two molecules resulting in a change in ORR values. In this study, we investigated the variations in ORR values of NADH/FAD mixtures dissolved in tris(hydroxymethyl)aminomethane, phosphate buffer, and deionized water environments. In vitro solutions were prepared in various concentration ratios and the experimental and theoretical ORR values were obtained from fluorescence and absorption spectra in time series. Based on the spectroscopic analysis, we concluded that the inner filter effect causes an instant decrease in FAD fluorescence just after dissolution and that the oxidation-reduction coupled with oxygenation reaction results in time-varying decreases in NADH fluorescence and FAD emission.