This work optimized the novel biotransformation process of 4'-demethylepipodophyllotoxin (DMEP) into 4'-demethylepipodophyllic acid (DMEPA) by Bacillus fusiformis CICC 20463. Firstly, the biotransformation process was significantly affected by medium composition. 5 g/L of yeast extract and 10 g/L of peptone were optimal for DMEPA production (i.e., 2.81 + or - 0.21 mg/L), while not beneficial for the cell growth of B. fusiformis. This indicated that the biosynthesis of DMEPA was not corresponded well to the cell growth of B. fusiformis. 40 g/L of sucrose was optimal for DMEPA production (i.e., 2.94 + or - 0.17 mg/L), and 3 g/L of NaCl was the best for DMEPA production (i.e., 4.10 + or - 0.18 mg/L). Secondly, the production of DMEPA was significantly enhanced by the control of substrate concentration and culture pH. 100 mg/L of substrate was optimal for DMEPA production (i.e., 6.47 + or - 0.35 mg/L), and DMEPA concentration was enhanced to 38.78 mg/L by controlling culture pH at 9.0 in the stirred-tank bioreactors. The fundamental information obtained in this study provides a simple and efficient way to produce DMEPA by biotransformation.