A set of experiments have been performed to investigate the production of biobutanol as a novel applicable biofuel in a bioelectrolysis cell (BEC). The objective of this work was to understand the mechanism and production rate of the biobutanol by bioelectrosynthesis (BES) using glucose as a substrate. Four main factors, such as electrode materials, substrate concentration, operating temperature, and poised applied voltage were investigated in batch mode to achieve optimum condition for producing maximum butanol by C. pasteurianum in BEC. Standard modified P2 medium (MP2) and standard minimal medium (SMM) were used as fermentation media in batch operation mode. Numerical optimization using central composite design (CCD) method has been used to maximize the butanol production within the experimental range. The maximum butanol production 13.31 g/L was obtained by applying 1.32 V indicating the suitability of this procedure. The results showed that by applying optimum conditions in SMM, the butanol could be enhanced remarkably by electroactive microorganisms in cathode chamber.