In order to enhance the production of laccase and reduce the cost of laccase production, the culture medium for the growth of Coriolus hirsutus, a laccase production strain, was studied using molasses distillery wastewater (MDW) as carbon source. The different concentrations of molasses distillery wastewater and proportion of urea were optimized consequently through Box-Behnken experiment design under the foundation of signal factor experiment. As the optimization result, the production of laccase with the concentration of MDW 47% and proportion of urea 0.5% was six times higher than that from the original medium used previously. This indicates that the laccase production using MDW as carbon source is feasible. At the same time, the study makes the theoretical foundation for the exploitation and utilization of MDW to produce high value product.