Abstract: On the basis of establishing the analysis method of indigo content, a new and efficient method for the preparation of indigo with high purity was established, and the antibacterial activities of different crude indigo extracts and high purity indigo were studied. Using Baphicacanthus cusia leaves as raw material, three crude extracts samples 1-3 with different indigo mass fractions(3.06%, 20.13%, 22.16%) were obtained by water extraction, stir with alkali, acid neutralization and sodium hydroxide for impurity removal. On this basis, single factor test and Box-Behnken response surface design method were used to optimize glucose reduction process for acquiring high purity of indigo sample 4. Finally, oxford cup method was employed to compare the antibacterial activities of three kinds of crude indigo extracts and high purity indigo against Aspergillus niger, Staphylococcus aureus and Escherichia coli. Results showed that the optimal conditions of glucose reduction were glucose concentration of 25 g/L, sodium hydroxide concentration of 26.9 g/L, reaction temperature of 60 ℃ and reaction time of 30 min. Under the optimum conditions, the purity of indigo could reach 96.87%(sample 4), and the yield of indigo was 76.88%. Results of bacteriostatic experiment demonstrated that the three kinds of indigo crude extracts and high purity indigo had inhibitory effect on A. niger, S. aureus and E. coli, and the inhibitory effects on the three kinds of bacteria increased with the increase of the mass fraction of indigo in the samples. Moreover, the inhibition zone diameter, MBC values, MIC values and IC₅₀ values of these extracts were 9.00-19.69 mm, 1-512 mg/L, 8-64 mg/L(A. niger) and 0.6-448 mg/L, respectively. The high purity indigo had a good inhibitory effect on A. niger, and its MIC, MBC and IC₅₀ values were 1, 8 and 0.6 mg/L, respectively.