Traditional fermentation is carried out initially fermented by natural microorganisms present in the substrate. This usually leads to a long fermentation time and occasional failure in the fermentation, and creates inconsistencies in the product quality. Teff injera is one of the widely used Ethiopian fermented foods that suffer such problem. Therefore, the aim of this study was the identification and isolation of dominant species of yeast and lactic acid bacteria for the optimizations of the traditional fermentation process of teff injera dough fermentation. Fermented teff dough sample was plated on potato dextrose agar and de Man, Rogosa & Sharpe agar at 24 hours intervals of different fermentation time from 0 to 96hrs for the enumeration of LAB and yeast respectively. The dominant of LAB and yeast species were identified and isolated by using morphological, physiological, and biochemical characterization. 500g of sterilized teff flour was fermented with each of the identified and isolated single starter cultures (LAB and yeast) with different inoculum levels (0.5, 0.75, and 1ml) at 0, 24, 36, and 48 hrs of fermentation time. The growth kinetics of both LAB and yeast in fermented teff dough was increased significantly (p<0.05) from 8.91 to 9.97 logcfu/gm and 5.98 to 7.62 logcfu/gm. In this study, five lactic acid bacteria (LAB) and three yeast dominant strains were identified. The LAB strains were identified as Lactobacillus fermentum (26.7%), Lactobacillus brevis (20%), Lactobacillus plantarum (13.3%), Bacillus subtilis (20%), and Enterococcus casseliflavus (20%), and the yeast species included Saccharomyces cerevisiae (40%), Candida krusei (40%), and Pichia kudriavzevii (20%). There was a significance difference (p≤0.05) in pH and TA of fermented teff dough and teff injera due to different single starter culture, fermentation time, and inoculum size. Regarding the sensory acceptability, the panelist preferred that fermentation sample with 1 ml inoculums of saccharomyces cervicea at 24 hours having the best color, taste, aroma, and rollability with the score of 4.39, 4.05, 4.24 and 4.18 whilst sample fermented with Pichia kudriavzevii was preferred in terms of top and bottom surface, eye uniformity, overall acceptability with a score of 4.34, 4.37, and 4.21 compared with the control (teff injera without starter culture the control at p<0.05. Simultaneously, the panelist preferred that fermentation sample with 0.5 ml inoculums of saccharomyces cervicea at 36hrs hours having the best texture. As a conclusion, the application of single starter culture for the teff dough fermentation was decreased fermentation time with a rapid increase of acid production. Hence, further study will require on molecular characterization of the isolated LAB and yeast by using 16s rRNA to confirm the isolate as LAB and yeast. Keywords: - Dominant, Fermentation, Identification, LAB, Species and Yeast