In this study, Ponceau S dye, which is one of the hazardous dyes found in industrial wastewater, was examined for hydrogen production in single chamber-free membrane-free microbial electrolysis cells at different concentrations (10-40mg L-1). A gas content analysis (hydrogen, carbon dioxide, and methane) was measured daily using gas chromatography to determine the effects of the Ponceau S on hydrogen production levels. Hydrogen was successfully produced in the presence of Ponceau S dye, but the gas production levels were affected by the concentrations of Ponceau S. The maximum hydrogen production was measured as 18mL at a concentration level of 20mg L-1. Decolorization ratios of Ponceau S were in the range of 85-100%. Hydrogen production rates increased in the presence of Ponceau S (20mg L-1); however, yield (%) of the production decreased when compared to the control group. The percentage of COD removal was 94.78% in the presence of 40mg L-1 of Ponceau S. In conclusion, hydrogen can be generated using wastewaters contaminated with azo dyes such as Ponceau S, and decolorization of the dye can be achieved, simultaneously.