The strains with strong nitrogen fixation ability were isolated from the burdock root by improved Ashby nitrogen free medium and acetylene reduction method, and identified by morphological observation and molecular biology techniques. The culture conditions of the strain were optimized by response surface methodology. The results showed that a strain numbered NF-112 with strong nitrogen fixation ability was screened and preliminarily identified as Klebsiella pneumoniae. The optimal culture conditions of the strain were obtained as follows: initial pH 5.3, culture temperature 28 ℃, glucose addition 26 g/L. Under the optimal culture conditions, the OD600 nm value was 1.76. The study laid a theoretical and technical foundation for the special bio-organic fertilizer preparation of the medicinal plant burdock.