The annual output of tobacco in China is about 3 million tons, accounting for one third of the world's total output, of which about 25% of the tobacco leaves, tobacco end and other leftovers can not be reasonably utilized. Reasonable development and utilization of tobacco waste resources will have good economic and environmental benefits. Hence, in this work, the potential of photofermentative hydrogen production from different parts of tobacco leaves was evaluated. The upper, middle and stem of tobacco leaf were used as substrates to study the hydrogen production in the process of bio hydrogen production. Bio-hydrogen yield capacity and energy conversion efficiency were taken as the reference.The middle part of tobacco leaf was the most suitable substrate for photo fermentation bio hydrogen production. Under the conditions of light intensity of 3000lux, substrate concentration of 33.34g/L, initial pH value of 7.00 and bacterial inoculation amount of 30%, the maximum cumulative hydrogen production was about 106mL / g.Specific hydrogen yield was higher than the control group and Highest energy conversion efficiency was obtained.