This study explored the interrelationship between nano-TiO₂ addition and photo-fermentative hydrogen production (PFHP) of corn straw. The maximum cumulative hydrogen volume (CHV) was up to 688.8 mL under the optimal photo-fermentative process conditions with nano-TiO₂ addition of 300 mg/L. Initial pH and interaction between substrate concentration and light intensity had highly significant effects on PFHP of corn straw with nano-TiO₂ addition. With the improvement of CHV, nano-TiO₂ addition decreased the optimal initial pH and substrate concentration for PFHP of corn straw. Moreover, nano-TiO₂ addition promoted the metabolism of butyric acid and acetic acid by photosynthetic bacteria HAU-M1, and significantly reduced the total concentration of intermediate byproducts during hydrogen production to a low level of 1.6–2.5 g/L, thus making the CHV, maximum hydrogen production rate (HPR) and average hydrogen content (HC) increased by 32.6%, 27.9% and 8.3% respectively over the control without nano-TiO₂ addition.