High-gravity fermentation, used for ethanol production from sugarcane, corn, and mixed substrates, offers several benefits. Yeast, a rapidly multiplying unicellular microorganism, can be adapted for high sugar and ethanol tolerance on a lab scale. However, different substrates can enhance fermentation efficiency. Our study consisted of two experiments. In the first, we compared simple batch feeding with a fed-batch system for yeast selection in high-gravity fermentation. We ran eight cycles with increasing initial sugar contents (50 to 300 g L−1). No significant differences were observed in the first seven cycles, but in the eighth, the fed-batch system showed lower glycerol and fructose contents and higher cell viability than the simple batch system. In the second experiment, we used the fed-batch system with 300 g L−1 from sugarcane, corn, and mixed wort. The results showed that mixed wort produced higher ethanol contents and greater fermentation efficiency compared to corn and sugarcane as substrates. In conclusion, our findings indicate that the fed-batch system is more suitable for high-gravity fermentation on a lab scale, and the combination of sugarcane juice and corn can enhance fermentation efficiency, paving the way for integrating these substrates in industrial ethanol production.