2D spectrofluorometry produces a large volume of spectral data during fermentation processes with recombinant E. coli, which can be analyzed using chemometric methods such as principal component analysis (PCA), principal component regression (PCR) and partial least square regression (PLS). An analysis of the spectral data by PCA results in scores and loadings that are not only visualized in the score-loading plots but are also used to monitor the fermentation processes on-line. The score plots provided useful qualitative information on four fermentation processes for the production of extracellular 5-aminolevulinic acid (ALA). Two chemometric models (PCR and PLS) were used to examine the correlation between the 2D fluorescence spectra and a few parameters of the fermentation processes. The results showed that PLS had slightly better calibration and prediction performance than PCR.