Natural melanin is of great potential value and application in the fields of pharmacology, cosmetics, and functional foods. In the present study, statistically designed experiments were conducted for the optimization of the media to enhance the production of melanin by submerged culture of Auricularia auricula. Glucose, tyrosine, peptone, and CaCO3 were found to have significant effects (P < 0.015) on melanin biosynthesis by a Plackett-Burman experimental design and subsequently optimized using response surface methodology. Optimal media were obtained at the following concentrations: glucose, 0.90 g/L; tyrosine, 6.68 g/L; peptone, 6.99 g/L; and CaCO3, 6.75 g/L. The validity of the optimum media was verified in separate experiments in which the melanin yield of 1008.08 mg/L was obtained under optimum conditions, compared with 306.52 mg/L at other conditions, i.e., a 3.29-fold increase. Furthermore, the important physical and chemical properties of A. auricula melanin were determined. The findings from the present study indicate that large-scale production of natural melanin by submerged culture of A. auricular could be a useful approach.