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For photo-hydrogen production offers the possibility of being renewable and carbon neutral, it has promising prospects in the clean energy future. The microorganisms from several natural sludge pools where has plenty of sunlight and with high organic load were used as samples. The mixed culture of photosynthetic bacteria (PSB) was cultivated by using selective medium method. Bacteria partial reflux device was employed for screening and optimization of mixed culture. The hydrogen production characteristics of mixed culture of PSB using cattle manure wastewater were also investigated. The results showed that, the mixed PSB culture grew fast and stable, and the conditions for its growth and hydrogen production were less strict than that of pure PSB. The optimum conditions for bacterial reflux were cell reflux time of 36h and cell reflux volume of 30%. The maximum hydrogen production rate was 28.3mL/(L·h), and mean concentration of H2 was 55%. When using cattle manure wastewater as substrate for biohydrogen production by mixed PSB, the hydrogen production duration was up to 216h, the average hydrogen production rate was 11.65mL/(L·h), substrate utilization ratio was 71.48%, and the average substrate conversion rate was 52.60mL/g. 