Abstract: Bilirubin oxidase (EC.1.3.3.5) produced by Myrothecium verrucaria shows a promising potential in the application of environmental protection. In order to increase the production of bilirubin oxidase by M. verrucaria, we investigated the effects of carbon, nitrogen, metal ions and inducers using the single-factor and orthogonal design methods. The results show that glucose and soya peptone are the best carbon and nitrogen sources, respectively. A higher enzyme activity was achieved with the addition of 2 mmol/L Cu2+ and 0.1 mmol/L of inducers. The orthogonal design experiment showed that the optimal medium was glucose 10 g/L, soya peptone 7.5 g/L, CuSO4 1 mmol/L and gallic acid 0.1 mmol/L. The enzyme activity was enhanced three times under this optimal medium. The optimal temperature for the enzyme activity was 40℃, and optimal pH was 7.5. With an enzymatic activity of 1 000 U/L, the initial purified bilirubin oxidase could decolorize more than 90% of indigo carmine (100 mg/L) within only 50 min, suggesting a broad application prospect of bilirubin oxidase in dye effluent decolorization.