Abstract: Abstract: A microbial challenge test was proposed to optimize the sterilization process for high efficiency of packaging materials in roof-carton aseptic filling. Bacillus stearothermophilus spores were evenly coated on the inner surface of the clean roof-carton packaging materials. A filling machine was then used to sterilize the packing material. The plate counting and the Most Probable Number (MPN) method were selected to recycle the residual spores on the surface of the sterilized material. The sterilization efficiency was characterized by the Log Cycle Reduction (LCR) of inoculated spore. A single factor test was carried out to determine the effects of hydrogen peroxide solution concentration, vaporized hydrogen peroxide vapor temperature, air pressure of vaporization heat, and UV irradiation time on the sterilization efficiency. A response surface test was designed for the significant variable conditions, according to the Box-Behnken principle. As such, a quadratic polynomial regression model was established for the sterilization efficiency of packaging materials. The aseptic filling machine was normally required that the sterilization effect of packaging materials was up to a 4-lg reduction in the number of inoculated microorganisms. The LCR value was calculated as 5 using the MPN statistical method. The optimal process parameters were determined to consider the comprehensive influencing factors, such as the design cost of mechanical structure, tolerance temperature of packaging materials, and hydrogen peroxide consumption. A concentration test of hydrogen peroxide was also utilized to detect the residual concentration in the package after sterilization. A hot-air drying was then used to adjust the process parameters for the standard residues of hydrogen peroxide. The results show that the sterilization efficiency of packaging materials in the filling machine dramatically improved with the increase of the parameters, among which the concentration of hydrogen peroxide solution, the steam temperature of hydrogen peroxide, and ultraviolet radiation time posed a significant effect on the sterilization efficiency, whereas, the vaporization heat air pressure was no significant effect on the sterilization efficiency. Therefore, a quadratic polynomial regression of sterilization efficiency was obtained from the response surface test, where the concentration of hydrogen peroxide solution, the temperature of hydrogen peroxide vapor, and UV irradiation time were selected as the variable conditions. The significant order of sterilization efficiency was then ranked as the hydrogen peroxide solution concentration > hydrogen peroxide steam temperature > ultraviolet irradiation time. Once the LCR sterilization of packaging material was required as 5, the optimal process parameters were the 8% hydrogen peroxide solution concentration, and 230 ℃ hydrogen peroxide vapor temperature. The 104 CFU spore-inoculated roof carton sample was tested 20 times, and the packaging material was completely sterilized, indicating the reliable regression model for the influence of various factors on the LCR value. This finding can provide the technical basis for the sterilization process of packaging materials during aseptic filling.