Adenoviruses are the most commonly used vectors in clinical trials for gene therapy. How to efficiently produce abundant and high-quality adenoviral vectors for therapeutic research is a challenge for biochemical engineers. A recombinant adenovirus carrying a green fluorescent protein (GFP) gene together with an anchorage-dependent 293 cell line is used as a model system for evaluating the effects of chemicals on adenoviral production in this study. Our aim is to develop a formulation to be added to an infection medium that could enhance the in vitro production of adenoviral vectors. Eleven ingredients obtained from a literature survey were screened for their stimulatory effects on adenoviral production using the 50% tissue culture infectious dose (TCID50) method. Among these ingredients, sucrose and mannitol when supplemented to the infection medium significantly increased adenovirus titer. Central composite design and response surface methodology were also adopted to determine the optimal concentrations of sucrose and mannitol. The formulation developed, which is composed of DMEM/F12 medium plus 0.54 M sucrose and 0.37 M mannitol, can significantly increase adenoviral production by 13-fold that of the control (DMEM/F12 medium).