The relevant fluorophores in cells can be a key component to understanding cellular activities, which in turn explains states of cultivation processes. The autofluorescence inside microorganisms can be measured by 2D fluorescence spectroscopy which is an effective and non-invasive device for an on-line monitoring of bioprocesses. We detected the following intrinsic fluorophores which are part of metabolic pathways for yeast growth during fermentation in real-time: tryptophan, pyridoxine, NADH, riboflavin, FAD, and FMN. The changes of these intrinsic fluorophores were observed from the yeast cultivations under three conditions: (i) normal batch, (ii) glucose addition during glucose growth phase, and (iii) glucose addition during ethanol growth phase after a diauxic shift. The glucose addition during ethanol growth phase demonstrated the correlative changes of the fluorophores, which was a key component in the metabolic switch from ethanol to glucose growth phase. Additionally, the quantification of conversion between tryptophan and NADH was shown as a proportional factor. It was calculated from the ratio of the area of NADH and tryptophan fluorescence intensity after the glucose addition until depletion. The proportional factor was independent on various glucose concentrations with the coefficient of determination, R-2 = 0.999.