Abstract: This research aimed to enhance biohydrogen production efficiency from maize stover via photo-fermentation. Anthraquinone-2-sulfonate (AQS) was also introduced into the hydrogen production. Among them, the AQS functioned as an electron shuttle to maintain the reducing environment for the interspecies electron transfer. A systematic evaluation was conducted to explore the effects of AQS concentrations on hydrogen yield, energy conversion, and metabolic pathways. The optimization mechanism of redox potential was also obtained after photofermentation. A series of experiments was performed in 150 mL conical flasks with 5 g maize stover, 100 mL citrate-buffered medium, 2 mL cellulase, and different AQS concentrations (0-150 mg/L). The pH was adjusted to 7.0 after inoculation with 50 mL HAU-M1 photosynthetic bacterial consortium. The sealed systems were then incubated at 30 °C under 3000 lux illumination. The key indicators were selected as the cumulative hydrogen production, hydrogen production rate, pH, oxidation-reduction potential (ORP), reducing sugar content, and dissolved organic matter (DOM) content. These parameters were monitored at 12-hour intervals and then analyzed using Origin 2022, thereby evaluating hydrogen production performance and energy conversion efficiency. Triplicate runs were conducted for each condition. The results showed that the addition effectively maintained the reducing environment to significantly regulate the photobiological hydrogen production. Once the concentration was within the range of 30 to 90 mg/L, the hydrogen production performance of photo-fermentation increased with the increasing concentration. While there was a gradual decrease within the range of 120 to 150 mg/L, the concentration increased. The supplementation of 90 mg/L AQS was the most effective in improving the hydrogen production of photofermentation. The cumulative hydrogen production of maize stover reached a maximum of 359 mL at this concentration, and the energy conversion rate reached 5.63%. Compared with the control group, the cumulative hydrogen production and the energy conversion rate increased by 15.06%. An optimal amount of AQS reduced the ORP of the photo fermentation for the strong reductive potential. Meanwhile, the reducing property was enhanced with the increase of the amount; The pH value of the treatment group was always lower than that of the unadded control group, which was attributed to the decomposition of organic matter in the process of fermentation. The main by-products, volatile fatty acids (VFAs), significantly reduced the pH value of the system. The DOM in the photo-fermentation broth consisted of soluble microbial metabolites and humic acid-like substances. The DOM fluorescence peak intensity in the experimental group with the addition of AQS was 26.6% lower than that of the control group. The addition of AQS at the optimal concentration effectively reduced the production and accumulation of the metabolic intermediates of tryptophan-like and humic acid-like substances. The AQS functioned as a redox electron mediator. The coupling efficiency of the substrate reaction with the electron transfer chain also prompted more carbon sources to flow directly into the hydrogen-producing metabolic pathway rather than toward the DOM generation. A mechanism of ‘precursor competition and product inhibition’ existed between DOM generation and the photo-fermentation hydrogen production. The main inhibition mechanisms included complexation of humic acid with nitrogenase cofactor, inhibition of ATP synthesis, and the decreased efficiency of the electron transport chain. The activity and hydrogen production efficiency of photosynthetic bacteria were inhibited in experimental groups at higher concentrations of AQS (120 and 150 mg/L). The high concentration of quinone redox mediators inhibited the nitrogenase activity and biomass production of the photosynthetic bacteria, damaged their cell membranes, and led to their death, which significantly reduced the photo-fermentative hydrogen production. As such, the AQS enhanced both the yield and biohydrogen production rate from maize stover, where the reducing property was improved by electron-conduction performance. The AQS was achieved in a higher energy conversion rate (5.63%) than Fe²⁺ (5.21%) and phosphate (3.2%). The superior cost-effectiveness was obtained, compared with 6.3% (with 150 mL/g Graphene Oxide at 1980 Yuan/g versus 90 mg/L AQS at 1.76 Yuan/g). These findings can provide a sustainable solution to enhance the biomass photo-fermentation performance. A strong reference can offer to maintain the reductive activity for the high biohydrogen production efficiency from maize stover.