The optimal conditions for melanin extraction from Auricularia auricula-judae (Hei 29) fruiting bodies was determined on the basis of the extract yield of melanin, calculated by using a single-factor experiment and response surface methodology. Its antioxidant activities were also studied in vitro. Various optimal process conditions for melanin extraction were determined by using Design-Expert software: incubation temperature, 69.11°C; incubation time, 58.66 minutes; and incubation pH, 12.81. Under these conditions, the melanin yield was 2.59%. We found that the antioxidant activities of A. auricula-judae melanin in vitro were strong against DPPH radicals and superoxide anions. The rate of DPPH radical scavenging was 63.04% when the A. auricula-judae melanin concentration was 0.36 mg/mL; the rate of superoxide anion scavenging reached 39.79% when the concentration was 0.375 mg/mL. However, the antioxidant activity against hydroxyl radicals was somewhat weak; the rate of scavenging reached only 7.47% when the A. auricula-judae melanin concentration was 0.06 mg/mL.