Optimization of three parameters: agitation rate (A; 100, 200 and 300 rpm), aeration rate (B; 0.5, 1.5 and 2.5 vvm) and aeration timing (C; 2, 4 and 6 h), for ethanol production from sweet sorghum juice under very high gravity (VHG, 290 g L−1 of total sugar) conditions by Saccharomyces cerevisiae NP 01 was attempted using an L9 (34) orthogonal array design. The fermentation was carried out at 30 °C in a 2-L bioreactor and the initial yeast cell concentration was approximately 2 × 107 cells mL−1. The results showed that the optimum condition for ethanol fermentation should be A2B3C2 corresponding to agitation rate, 200 rpm; aeration rate, 2.5 vvm and aeration timing, 4 h. The verification experiments under the optimum condition clearly indicated that the aeration and agitation strategies improved ethanol production. The ethanol concentration (P), productivity (Qp) and ethanol yield (Yp/s) were 132.82 ± 1.06 g L−1, 2.55 ± 0.00 g L−1h−1 and 0.50 ± 0.00, respectively. Under the same condition without aeration (agitation rate at 200 rpm), P and Qp were only 118.02 ± 1.19 g L−1 and 2.19 ± 0.04 g L−1h−1, respectively while Yp/s was not different from that under the optimum condition.