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To realize photo-biological hydrogen production under continuous flow mode, a groove-type flat-panel photobioreactor (GFBR) with high specific surface area was developed by carving groove on the surface of high-light transparent medium. With photosynthetic bacteria attached on the groove-type surface and the generated mature biofilm, cell-immobilization was finished within GFBR. To improve the performance of hydrogen production in GFBR, operating parameters related to substrate were comprehensively investigated using response surface methodology of Box-Behnken with three factors and three levels. The results showed that the significant degree of single factor on hydrogen production rate of GFBR was followed as hydraulic retention time, initial pH value and initial substrate concentration. While, interaction effect between initial pH value and initial substrate concentration on hydrogen production rate of GFBR was also significant. The optimum hydrogen production rate of 2 242mmol/(L·h) was attained with operating parameters of initial substrate concentration of 54.3mmol/L, hydraulic retention time of 21.5h and initial pH value of 7.1.