Effluents loaded with various synthetic dyes are considered as a huge burden to the surrounding ecosystems. Sudan dyes are relatively difficult to decolorize due to its water-insolubility. In the present study, the strain Shewanella putrefaciens CN32 was firstly applied to decolorize Sudan dyes under the anaerobic condition, and the physicochemical parameters on the decolorization were optimized. The results demonstrated that the suitable decolorization condition was temperature 26 °C, initial pH 7.0-8.0 and NaCl concentrations 0-20 g/L. Electron competitive acceptors including nitrite, nitrate, dimethyl sulphoxide and oxygen could cause the significant inhibition to the decolorization of Sudan dyes. Biosurfactant rhamnolipid played a positive role in enhancing the decolorization of Sudan I. The co-culture of S. putrefaciens CN32 and Bacillus circulans BWL1061 is reported for the first time to accelerate the decolorization through improving the synergistic effect of enzymatic degradation and biological reductive effect. The highest decolorization of 90.23% to Sudan I was achieved within 108 h, suggesting that co-culture technique has a good potential in the treatment of dyeing wastewater. Furthermore, the microbial toxicity tests indicated that the toxicity of Sudan I to Escherichia coli BL21 and Bacillus subtilis 168 was obviously decreased after the decolorization.