Abstract: Research on microbial activity of photosynthetic bacteria in the process of hydrogen production plays an important role in revealing the nature of hydrogen production and increasing the efficiency of hydrogen production by photosynthetic bacteria.The effects of glucose as carbon substrate on microbial activity of photosynthetic bacteria were researched in this paper,the cause of cell growth activity decline and the influence of glucose on the absorption spectra of photosynthetic bacteria were also analyzed. It showed that in the process of photosynthetic bacteria utilizing glucose to produce hydrogen, glucose was metabolized into organic acid which leads to pH value decreased, and the cell growth activity was inhibited by low pH value. There was no hydrogen produced When the added concentration of glucose below 3 mmol/L, however, it was apparent that the characteristic peaks of absorption spectra and the micrpbial activity was promoted by substrate. When the concentration of glucose was added up to 30 mmol/L, the hydrogen production activity increased significantly, but the characteristic peaks of absorption spectra decreased obviously, and the growth activity was inhibited by substrate. photosynthetic bacteria utilized glucose as substrate to produce hydrogen, the best added concentration of glucose was 180 mmol/L.