Abstract: Objective: In order to expand the research of functional products, the content of active ingredients in Pueraria thomsonii was increased by microbial fermentation technology. Methods: Firstly, the fermentation process of Pueraria thomsonii was optimized by the combination of single factor test and response surface methodology with the decoction pieces of Pueraria thomsonii as raw material, L. acidophilus as fermentation strain, puerarin content as index, and inoculation amount, solid-liquid ratio, fermentation time and fermentation temperature as acting factors. Secondly, the contents of puerarin, daidzin, daidzein, total isoflavones, soluble polysaccharides and total starch in Pueraria thomsonii were determined by high performance liquid chromatography, ultraviolet spectrophotometry and dual wavelength method before and after fermentation. Results: The optimal fermentation conditions were as follows: Inoculation amount 5%, solid-liquid ratio 1:3 g/mL, fermentation time 36 h, fermentation temperature 29 ℃. The relative error was 0.29% between the actual content 8.1854 mg/g and predicted value 8.2092 mg/g of puerarin. After fermentation, the contents of puerarin, daidzin, daidzein, total isoflavones, soluble polysaccharides and amylopectin in Pueraria thomsonii increased, while the contents of amylose and total starch decreased, showing significant differences compared with those before fermentation (P<0.05). Conclusion: The results showed that microbial fermentation technology could improve the content of active ingredients in Pueraria thomsonii.