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 $195.3m{ell}{cdot}H_2/{ell}$ broth for Rhodobacter spaeroides KCTC1425 and $271.8m{ell}{cdot}H_2/{ell}$ 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 Y J-1, the optimum mixing ratio of hydrogen production was 20 and 80. Adding volume of yeast extract for maximum hydrogen production was 15 $g/{ell}$, but there was no effect over that. $Na_2MoO_4$ was most effective among the inorganic salts, and the optimum volume was 0.4 $g/{ell}$. In semi-continuous culture, total hydrogen production was $13086m{ell}{cdot}H_2/{ell}$ broth for 144 h with operating period of 24 h.