Sorghum is a remarkably nutritious cereal plant primarily found in Africa. It was introduced to Indonesia in 1989. Joining the effort to improve the quality of local flour as part of the Indonesian government’s food security program, we applied the yeast Saccharomyces cerevisiae during sorghum flour fermentation with different parameters to yield better final flour products. Since low digestibility of proteins and high levels of tannins that are present in grains are considered as obstacles to favourable sorghum flour production, we aimed at formulating a method for flour production by using the mutant sorghum variety called Samurai 2, and employing various fermentation periods and concentrations of S. cerevisiae as starter culture. The quality controls were performed on the basis of chemical and physical properties. The present work employed a complete factorial randomised design, by varying the durations of fermentation (20, 40, and 60 h) and starter culture concentrations of 107 CFU/mL (0, 2, 4, and 6%, w/v). The results showed that the longer the fermentation time and the higher the starter concentration, the lower the water, ash, and in-flour tannin contents, whiteness intensity, and viscosity (p < 0.05). On the other hand, this treatment increased the levels of dissolved protein (p < 0.05), and the microstructure of starch granules became coarser.