Background. Astragalus was a well-known traditional herbal medicine, widely used in human s , livestock and poultry in China and E ast Asia. Fermentation could improve health-promoting biological substance by probiotics. Methods. We investigated Astragalus that was fermented using probiotics including Enterococcus faecium , Lactobacillus plantarum and E nterococcus faecium + L actobacillus plantarum and applied the PacBio single molecule, real-time sequencing technology (SMRT) to evaluat e the quality of Astragalus fermentation production. Results. We found the production rates of acetic acid, methylacetic acid , ethylacetic acid and lactic acid using E. faecium + L. plantarum fermentation were 1866.24 mg/kg on day 15 , 203.80 mg/kg on day 30 , 996.04 mg/kg on day 15 and 3081.99 mg/kg on day 20 , respectively. Other production rates were: polysaccharide s, 9.43%, 8.51% and 7 . 59% on day 10; saponins , 19.6912 mg/g, 21.6630 mg/g and 20.2084 mg/g on day 15; and flavonoid s, 1.9032 mg/g, 2.0835 mg/g and 1.7086 mg/g on day 20 using E . faecium , L . plantarum and E. faecium + L. plantarum , respectively. According to SMRT analysis of the microbial composition s of nine Astragalus samples, we found after fermentation on day 3 , E. faecium and L. plantarum became the most prevalent species. Moreover, E. faecium + L. plantarum gave more positive effects than single strains in the Astragalus solid state fermentation process. Inclusion. Our data have demonstrate d that the SMRT sequencing platform is applicable to assessing the quality of Astragalus fermentation.