Light has been considered as a key factor for hydrogen enhancement of photo-fermentation. This study proposed a strategy to improve the light conversion efficiency of photo-fermentative hydrogen production of Arundo donax L. by using light-dark duration. For this purpose, 6 projects (continues light, 24h light -24h dark, 24h dark- 24h light, 48h light-48h light, 48h dark-48h light, and continues dark) light disturbances were carried out to estimate the strategy. The effect of light-dark duration on cell growth of photosynthetic bacteria, hydrogen production, and light conversion efficiency were performed to test the strategy. Moreover, heatmap was novelty employed to analysis the correlation between light-dark duration with cell biomass yield, hydrogen yield, and light conversion efficiency.