Culture conditions for biological hydrogen production were investigated in wastewater of Takju manufacturing factory. Rhodobacter spaeroides KCTC1425, photosynthesis bacteria, and Enterobacter cloacae YJ-1, anaerobic bacteria were used. The hydrogen production were 195.3 ㎖？H2/ℓ？broth for Rhodobacter spaeroides KCTC1425 and 271.8 ㎖？H2/ℓ？broth for Enterobacter cloacae YJ-1 during 36 h. The hydrogen production increased with light intensity, and were highest over 12000Lux. In mixed culture of Rhodobacter spaeroides KCTC1425 and Enterobacter cloacae YJ-1, the optimum mixing ratio of hydrogen production was 20 and 80. Adding volume of yeast extract for maximum hydrogen production was 15 g/ℓ, but there was no effect over that. Na2MoO4 was most effective among the inorganic salts, and the optimum volume was 0.4 g/ℓ. In semi-continuous culture, total hydrogen production was 13086 ㎖？H2/ℓ？broth for 144 h with operating period of 24 h.