This study aimed to optimize the culture conditions of the termite-derived fungus Aspergillus chevalieri BYST01 for the production of physcion, a characteristic component of the traditional herb rhubarb, which has been commercially approved as a botanical fungicide in China. First, potato dextrose broth was screened as the suitable basal medium for further optimization, with an initial yield of 28.0 mg/L. Then, the suitable carbon source, fermentation time, temperature, pH value, and the rotary shaker speed for physcion production were determined using the one-variable-at-a-time method. Based on the results of single factors experiments, the variables with statistically significant effects on physcion production were further confirmed using the Plackett-Burman design (PBD). Among the five variables, temperature, initial pH, and rotary shaker speed were identified as significant factors (P < 0.05) for physcion productivity in the PDB and were further analyzed by response surface methodology (RSM). Finally, we found that the maximum physcion production (82.0 mg/L) was achieved under the following optimized conditions:initial pH 6.6, rotary shaker speed of 177 rpm, temperature of 28 °C, and glucose concentration of 30 g/L in PDB medium after 11 d of fermentation. The yield of physcion under the optimized culture conditions was approximately threefold higher than that obtained using the basal culture medium. Furthermore, the optimum fermentation conditions in the 5-L bioreactor achieved a maximal physcion yield of 85.2 mg/L within 8 d of fermentation. Hence, response surface methodology proved to be a powerful tool for optimizing physcion production by A. chevalieri BYST01. This study may be helpful in promoting the application of physcion produced by A. chevalieri BYST01 to manage plant diseases.