Continuous tests of dark and photo co-fermentative hydrogen production were for the first time conducted in an 40 L tubular multi-cycle bioreactor. adapting liquid baffle, the reactor specific surface area was improved and the reaction path was extended. Using multi-channel tube to enhance fluid flow properties. Using light-dark sub-zone reaction to ensure that the best condition of hydrogen-producing microbial. The continuous operation Parameter of hydraulic retention time(HRT) and organic loading rate(OLR) were evalutaed, and a 30 d continuous hydrogen production of reactor with Paulownia biomass was conducted. The result shows that In the process of continuous hydrogen production of the reactor for 30 days, the mean hydrogen production of 0.54 L/h, the average hydrogen concentration of 53.84%, and the mean raw material utilization rate was 89.93% in the condition of HRT 40 h, OLR 1.0 g/L•h,and light-dark alternative time 1:4. The overall parameters and operating indicators of bioreactor are stable with good operation stability.