Abstract: In order to study the resources of nitrogen-fixating organism from soils of Chinese herbal medicine in Gansu province, we isolated the microorganisms with nitrogenase activity from herbal rhizosphere soils of Codonopsis, Astragalus and Angelica, respectively; then purified and identified each isolated single strain by molecular biology; finally detected nitrogenase activity of each strain using acetylene reduction. The results showed that the three kinds of rhizosphere soils were with rich nitrogen-fixing organism resources, and 28 isolated strains were mostly identified as Rhizobium sp., Brevibacillus sp., Pseudomonas sp., Achromobacter sp. and Ensifer sp., with 53.5 C₂H₂ nmol·(h·ml)^-1~295.3 C₂H₂ nmol·(h·ml)^-1 of nitrogenase activity; and the nitrogenase activity of Achromobacter sp. was higher than the others. This study provides a theoretical basis for selecting bacteria to produce a highly effective nitrogen-fixing fertilizer using in herbal soils.