Abstract: OBJECTIVE To optimize the processing parameters of Indigo Naturalis by Box-Behnken response surface methodology. METHODS The yields of indigo and indirubin were taken as the response values respectively, Box-Behnken response surface design method was used to optimize the processing parameters of Indigo Naturalis based on the single factor methodology. Expert Design 8.0.6 software was used to analyze the data. RESULTS The optimal conditions for the orientation-generated indirubin predicted by the regression model were as follows:the material ratio was 1:10, the hydrochloric acid concentration was 3%, the O₃ time was 73 s, the extraction temperature was 79℃, the extraction time was 33 min, and the yield of the indirubin obtained under this condition was 0.758 mg·g^-1. The optimal conditions for the orientation-generated indigo predicted by the regression model were as follows:the material ratio was 1:10, the hydrochloric acid concentration was 3.4%, the extraction temperature was 79℃, and the extraction time was 33 min. The yield of indigo obtained under this condition was 2.07 mg·g^-1. CONCLUSION The study optimizes the processing parameters of the orientation-generated indirubin and indigo, and improves the quality of Indigo Naturalis.