Hydrogen is one of the most important energy sources in the world. The advantages of this sustainable energy source are numerous: it is clean, efficient, renewable, and nontoxic byproduct. Biological fermentation processes to produce hydrogen have been considered more than other processes. This project consists of main and supplementary sections. In the main section glucose and mineral medium were used as the substrate, similarly in the supplementary section fructose, sucrose, starch and mineral medium were used as the substrate. A microbial mixture was taken from anaerobic sludge unit of Isfahan waste water treatment plant. This mixture was used as an anaerobic culture. Ten different pretreatment operation that were used, consists of: 1) acidic (A), 2) basis (B), 3) heating (C), 4) cooling (D), 5) AB, 6) AC, 7) AD, 8) BC, 9) BD and 10) DC. In the A Pretreatment method, the highest amount of hydrogen was produced. Therefore, this method was used as a basis for later experiments. In the main experiment, three independent parameters of temperature, pH and the F/M (gCOD/gVSS) ratio were used. These parameters were used for evaluation and optimization of three dependent parameters of 1) hydrogen production rate (R [ml/h]), 2) efficiency of hydrogen production (Y [mlH2/gGlu]), and 3) efficiency of substrate degradation (S.D). Highest value of R, 6.55 ml/h, was obtained under these conditions: T=30.26 °C, pH=7.27 and F/M=1.788. Highest value of Y, 18.64 mlH 2 /gGlu, was obtained under these conditions: T=30.96 °C, pH=8 and F/M=0.25 and Highest value S.D, 98.8%, was obtained under these conditions: T=30.35 °C, pH=7.496 and F/M=0.25. Three volatile acids (acetic acid, butyric acid, and propionic acid) and ethanol were produced during the digestion process. In this process, butyric acid concentration was the highest and propionic acid was the lowest concentration. In the supplementary section, by chosen these conditions: T=31°C, pH=7.5 and F/M=0.25, all three responses R, Y and S.D were optimized. In the optimum condition, the supplementary testes for glucose, fructose, sucrose and starch were done similar to the main experiments. The response, R, was used to compare the results of supplementary and main testes. Responses, R, are in the order of glucose fructose sucrose starch. By comparing main experiment with supplementary for glucose at the optimal point, the amount of error between predicted value of R (5.65) and the experimental value of R (4.95) was 12.4%. This error is reasonable. Keywords: Hydrogen, Biogas, Anaerobic, Fermentation