Sugar wastewater contains of easily digestible substrate, i.e. glucose, and also rich in carbon and low of nitrogen content. Hydrogen production during sugar wastewater treatment by anaerobic microflora is being investigated. Objective of the research are to find an optimum operating parameter (pH, inoculums size, mixing speed and temperature) and determine performance of the process based on hydrogen production, volume of biogas and COD removal efficiency. The substrate was raw sugar wastewater, and the sludge containing microflora was taken from the clarifier of sugar wastewater treatment plant. Dark condition was applied and batch reactors of 10 ml working volume were used. Microflora in the sludge was inoculated at pH 5 to suppress methanogenic activities. No additional substrate was added to the inoculum. Preliminary experiment was conducted to determine optimum treatment time in terms of biogas production and COD removal, which was 60 hours. Initial pH was varied from 4 to 6, and inoculum sizes were 10%, 20% and 30%. Speeds of mixing were 100 to 250 rpm. The result shows that production of biogas was 0.5 ml to 5 ml per 10 ml of substrate at experiment at 200 rpm. The highest volume of biogas was obtained at pH 4 and 5 for 30% inoculum size. No biogas was produced at 150rpm and 250rpm. COD removal was found to be 5% to 822%, the highest removal was achieved at the experiment at pH 4, 20% inoculum size and 200 rpm. Hydrogen concentrations of the samples are being quantified.