Abstract: In order to optimize the fermentation substrate and technology of hamburger bread sourdough with the best inhibition effect on Penicillium, the single factor experiment combined with Box-behnken response surface experiment was performed to optimize the fermentation process. The bacteriostasis rate of sourdough to Penicillium was taken as evaluation index to select the optimal fermentation process such as starter and sugar type. Subsequently, the physicochemical indexes, microbial quantity, sensory, texture and shelf life of hamburger bread were determined under the optimal conditions. The optimum fermentation parameters were as follows: Lactic acid bacteria starter B and sucrose were added to sourdough, sucrose content 4.1%, fermentation time 20 h, and fermentation temperature 30℃. Under these conditions, the bacteriostatic rate of sourdough was 32.67%, compared with the theoretical predicted value, the relative error was 4.41%. The hamburger bread made under the optimal fermentation process has great antibacterial effect and physicochemical properties: Acid value 2.68 mg/g, specific volume 3.07 mL/g, peroxide value 0.15 g/100 g, hardness 1158.65 g, elasticity 0.88 mm. After the hamburger bread was cooled and packaged for 2 hours, the colony count and mold were not detected, and the shelf life was as long as 15 days at 20℃.