Probiotic beverages made from cereals are vital alternatives to the current growing drawbacks associated with dairy-based products. Accordingly, this study aimed to evaluate teff-based substrate for its applicable potential to delivering beneficial probiotics, Lactobacillus rhamnosus GG (LGG) and Lactobacillus plantarum A6 (LA6) with a view to develop fermented probiotic beverage. Also, two-dimensional fluorescence spectroscopy was applied to monitor the fermentation process, and the spectral data were analyzed using partial least squares regression (PLSR) and artificial neural network (ANN). The process parameters, time and inoculum were optimized to 15 h and 6 log cfu/mL, respectively. The 15 h fermentation of teff-based substrate, which was inoculated with 6 log cfu/mL mixed-strains of LA6 and LGG was run without pH adjustment. During fermentation, growth of LA6 and LGG increased to 8.416 and 8.247 log cfu/mL, respectively, which are above the minimum required counting of 6 to 7 log cfu/mL. Maltose and glucose were consumed during fermentation, and hence their amounts were decreased. However, lactic and acetic acids produced with fermentation time, therefore their concentrations were increased. Values of pH, titratable acidity, lactic acid, and acetic acids were measured between 6.3–3.9, 0.33–1.5 g/L, 0–1.7 g/L, and 0.04–0.231.5 g/L, respectively. Quantification of metabolites such as glucose and lactic acid with high performance liquid chromatography is challenging since it needs long time, high capital cost, and it is labor-intensive. Also, microbial counting with traditional methods such as plate counting technique is so tedious work. An alternative for this purpose would be the application of two-dimensional fluorescence spectroscopy (Masiero et al., 2013). It is an ideal instrument for the on-line supervision of fermentation processes. In addition, its measurement is non-invasive and does not have interference with the fermenting medium. The prediction abilities of PLSR and ANN models were evaluated by cross validation, and also their predicted values were compared with the actual measurements. This research findings verified that growth of LA6 and LGG, and concentrations of glucose and lactic acid could be predicted by two-dimensional fluorescence spectroscopy combined with PLSR and ANN. In conclusion, whole teff flour without supplement would be used as a substrate to produce probiotic beverage, and also the fermentation process could be supervised using two-dimensional fluorescence spectroscopy.