The gene encoding a putative triphenylmethane reductase (TMR)-like protein derived from Geobacillus thermoglucosidasius C56-Y593 (named as GtAZR) was synthesized, heterologously expressed in Escherichia coli, and extensively characterized for the first time. The recombinant GtAZR displayed its maximum activity at pH 5.5 and 40 A degrees C. GtAZR was stable at temperatures below 65 A degrees C. It also exhibited a broad pH stability and retained more than 90 % of its initial activities in pH range of 4.5-10.5 after incubating in various buffers for 1 h. Moreover, GtAZR showed significant stability against metal ions and organic solvents. GtAZR displayed broad substrate spectrum toward both azo and triphenylmethane dyes. As a sequence and structural TMR-like protein, GtAZR was characterized as an azoreductase biochemically due to its high specificity for azo dye rather than triphenylmethane dye. Molecular docking and mutagenesis analysis revealed that amino acids Asp-79 and Thr-80 are responsible for its azoreductase activity, which eliminated the steric hindrance caused by His-77 and Tyr-78 at the correspond sites in other structural homologous triphenylmethane reductase. The robust stability and substrate promiscuity of GtAZR made it a promising candidate for practical removal of mixed dye wastewater.