This study developed an artificial biofilm of *Rhodospirillum rubrum* bacteria immobilized within an alginate matrix using electrophoretic deposition (EPD) on an electrode. The resulting biofilm immobilized bacteria effectively and maintained a high survival rate, facilitating stable and high-efficiency hydrogen generation for longer periods compared to biofilms produced using free bacteria. Hydrogen production efficiency remained constant when the substrate was periodically replaced, indicating that the bacteria could survive within the biofilm for long-term hydrogen production. EPD produced mechanically stable large-scale biofilms economically and rapidly, which effectively overcame operational limitations such as culture medium temperature, pH, and flow rate. Therefore, this proposed method has the potential to accelerate the commercialization of biohydrogen production systems through large-scale biofilm production to facilitate continuous hydrogen generation. The technique can be utilized in various hydrogel-based applications, providing a cost-effective and efficient manufacturing process with customized biological and mechanical properties. The developed biofilms have implications beyond biohydrogen production and could be applied to hydrogel-based medical, cosmetic, and other fields.