[Objective] An indigo-producing gene was cloned and expressed, and its application in indigo production was investigated. [Methods] An indigo-producing gene from Burkholderia sp. IDO3 was cloned and heterogeneously expressed in Escherichia coli BL21(DE3). Recombinant strain was able to produce blue pigments. HPLC and LC/MS were applied to analyze the reaction product. The conditions for indigo production, such as rotary speed, temperature, and growth medium components were optimized, and indigo-producing curve was also determined under the optimal conditions. [Results] Recombinant strain IND_AB was successfully constructed and could produce blue pigment in LB medium. Product analysis revealed the blue pigment was indigo. Strain IND_AB could generate 22.9 mg/L indigo when incubated at 30 °C and 150 r/min. The indigo yield was improved to 25.4 mg/L and 35.1 mg/L after optimization of environmental parameters and medium components. The indigo yield was subsequently enhanced to 57.7 mg/L and 64.4 mg/L with the addition of 50 mg/L indole and 0.1 g/L tryptophan, which rose by 152.0% and 181.2% compared with initial yield. Time-course analysis indicated that there was no indigo production in the previous 6 h, and indigo was exponentially produced between 6?15 h and reached the maximum at 18 h. [Conclusion] Recombinant strain IND_AB could produce relatively pure indigo, and present study provided efficient genetic resource for indigo microbial production.