The use of biofuels helps to reduce the dependency on fossil fuels and therefore decreases CO2 emission. Ethanol mixed with gasoline in mandatory percentages has been used in many countries. However, production of ethanol mainly depends on food crops, commonly associated with problems such as governmental policies and social controversies. Sweet sorghum (Sorghum bicolor (L.) Moench) is one of the most potential and appropriate alternative crops for biofuel production because of its high biomass and sugar content, strong tolerance to environmental stress conditions and diseases, and wide adaptability to various soils and climates. The aim of this study was to select prospective varieties of sweet sorghum, optimum sowing times and densities to achieve high yields of ethanol production and to establish stable operational conditions in cultivating this crop. The summer-autumn cropping season combined with the sowing densities of 8.3–10.9 plant m−2 obtained the highest ethanol yield. Among cultivated locations, the soil with pH of 5.5 and contents of Al and Zn of 39.4 and 0.6 g kg−1, respectively, was the best condition to have an ethanol yield >5000 L ha−1. The pH ≥ 6.0 may be responsible for the significant reduction of zinc content in soils, which decreases both biomass of sweet sorghum and ethanol yield, while contents of N, P, K, organic carbon (OC) and cation exchange capacity (CEC), and Fe likely play no role. The cultivar 4A was the preferred candidate for ethanol production and resistant to pests and diseases, especially cut worm (Agrotis spp.).