Nowadays, microbial production of natural products represents a hot spot point in our environment and becomes an alternative way to chemical synthetic. In our research, Aspergillus flavus No. 3 shows great power in both biomass and kojic acid production. Plackett–Burman design utilize Egyptian sugarcane molasses as a sole carbon source giving kojic acid production of 0.82 (predicted 1.04) g/l to 24.65 (predicted 23.74) g/l, consuming sugar at 27.33 (predicted 26.96) % to 89.87 (predicted 87.46) % and forming dry biomass between 3.6 (predicted 3.8) g/l and 28.2 (predicted 28.05) g/l. The maximum kojic acid (24.65 g/l) and biomass value (28.2 g/l) obtained at 25°C; 9, 5 days of incubation, pH 3, 5; 0.5%, 2% inoculum size and the shaking rate at 150 rpm using fermentation medium (g/l) of sugarcane molasses, 60; yeast extract, 7, 3; KH₂PO_4, 2; ZnSO₄.7H₂O, 0.001 and MgSO₄.7H₂O, 1, respectively.