The mixed culture of photosynthetic bacterial was screened from activated sludge to carry out the experiment of hydrogen production. The effect of different nitrogen sources on the growth and hydrogen production of photosynthetic bacteria was studied, and the utilization pattern of nitrogen source in the process of hydrogen production by photosynthetic bacterial was also analysed. The results showed that: the photosynthetic bacteria had a significant selectivity in the process of growth to utilize nitrogen source. Inorganic nitrogen sources, especially ammonium salts was the most vulnerable to be used by photosynthetic bacteria, and organic nitrogen sources followed. Using (NH4)2SO4 as nitrogen source for growth, the best added concentration was obtined, which was 7 mmol/L. The maximum utilization appeared in 24~48 h of cultivated time, and the top consumption rate was 0.105 mmol/L. The impact of different nitrogen type on photosynthetic hydrogen production was not significant and the organic nitrogen was slighely better than inorganic nitrogen. When photosynthetic bacteria utilized (NH4)2SO4 as nitrogen source for hydrogen production and the added concentration was set as 3.5 mmol/L, the photosynthetic bacteria showed better activity of hydrogen production. The nitrogen source was only consumed during 0~48 h in the process of hydrogen production, and the cell no longer utilized the nitrogen source once the cell went into the period of hydrogen-producing peak.