The aim of this study was to evaluate the bioremediation capabilities of three kinds of periphyton (i.e. epiphyton, metaphyton and epilithon) immobilized in bioreactors to decolorize and biodegrade the sulphonated azo dye, amaranth. Results showed that periphyton dominated by phyla including Cyanobacteria, Proteobacteria and Bacteroidetes. Complete removal of dye was shown by all the biofilms periphyton (epiphyton showed highest removal efficacy) over a range of initial concentrations (50–500 mg L−1) within 84 h at pH 7 and 30 °C. Biodegradation of amaranth was confirmed through FTIR and HPLC and the biodegradation pathways were detected by GC–MS/MS analysis. The azo bonds in the amaranth were successfully broken by periphyton and amaranth was converted to non-toxic, aliphatic compounds including isobutene, acetyl acetate and ethyl acetate. The results showed the potential application of immobilized periphyton at industrial scale for the removal of azo dyes from wastewater containing azo dye amaranth.