5-HMF and furfural were found to be produced during the pretreatment and enzymatic hydrolysis stages of photo-fermentative hydrogen production. However, the roles of5-HMF and furfural on affecting the hydrogen yield are still unclear. This study aimed to explore the effects of 5-HMF and furfural addition on hydrogen production with photosynthetic bacteria. A single variable method was utilized to study the impact of photo-fermentation of biological hydrogen production by controlling the concentration of 5-HMF, furfural and their mixture. When 5-HMF was added, the inhibitory effect on photo-fermentation biological hydrogen production increased with the increase of concentration. When the concentration of furfural is 0.2g/L and 0.4g/L, it could promote hydrogen production compared to the control group (511.6mL), which was increased by 19% and 14%, respectively. and when the concentration of furfural is 0.8g/L, it had an inhibitory effect on hydrogen production. When the two substances were added with a ratio of 1:1 (addition amount was 0.2g/L), it would promote the photo-fermentation biological hydrogen production, while the hydrogen yield was inhibited when the addition amount further increased. When the concentration of the mixture reached 1.6g/L, the hydrogen production is 0, the growth and metabolism of photosynthetic bacteria were completely inhibited.