Indole is a common N-heterocyclic pollutant as well as a signaling molecule widespread in various environmental matrices. Several bacterial strains have been reported to be able to degrade indole, while the degradation capacity and functional enzymes are poorly documented. Herein, the degradation characteristics of a newly isolated indole-degrading strain Burkholderia sp. IDO3 were carefully investigated. Strain IDO3 exhibited superior degradation ability which could completely remove 100 mg/L indole within 14 h in mineral salt medium. It maintained comparable degradation performance under conditions of pH 4.0-9.0, temperature 25-35 degrees C and rotary speed 0-250 r/min, and most of the tested heavy metals and organic pollutants did not significantly affect the degradation process. Two important intermediates, i.e. isatin and anthranilate, were identified in indole degradation process. The genomic clone library technique with indigo-based screening method was successfully applied to screen the functional genes. Heterologous expression assay proved that recombinant E. coli strain carrying indole oxygenase and reductase genes iifCD could transform indole to indigo. Bioinformatic analyses indicated that the newly obtained enzyme lifC_IDO3 formed a phylogenetically separate branch from other related aromatic oxygenases. This study provides new insights into our understanding of indole degradation by Burkholderia strains and offers efficient bacterial resource for indole bioremediation.