This study aimed to evaluate the effect of light disturbance on the photo-fermentative hydrogen production of giant reed. For this purpose, different intensity light disturbances (4000lux,5000lux,6000lux,7000lux) at 24h and 36h during the photo-fermentative hydrogen production process were studied. The cumulative hydrogen yield, hydrogen production rate, OD660nm, pH, Oxidation-Reduction Potential (ORP), and total sugar yield were monitored to explore the influence in the study. The light disturbance has a significant effect on the photo-fermentative hydrogen production. The experiment was applied to the light intensity of 6000lux at 24h, achieved the highest cumulative hydrogen production capacity of 123.4 mL/g TS, which was 19.6% higher than that of the control group (110.4 mL/g TS) and 12.5% higher than the maximum level of the disturbance of 36h (104 mL/g TS). Under the best light disturbance conditions, the maximum hydrogen production potential (P) and the period of latency were 616.38mL H2 and 10.72 h. The timing supplemental illumination was proved to be a useful mean for enhanced hydrogen yield of photo-fermentation, which provided a new way for the efficient use of lignocellulosic biomass.